Retinopathy of prematurity
(ROP) is a
developmental disorder that occurs in the
incompletely vascularized retina of premature
infants and is an important cause of blindness in children in both the
developed and the developing countries. Progress in neonatal intensive
care in recent years has led to an increased survival of extremely low
birth weight (ELBW) infants weighing
≥1000 g at birth and,
subsequently, to an increasing incidence of ROP [1]. Lack of a screening
program in India has led to larger number of babies developing retinal
detachment, because the retina in ROP eyes does not seem to grow with
the growing eyeball [2]. It gives way at a vulnerable point because it
is stretched thin causing recurrent retinal detachment [3].
Re-attachment of retina also does not contribute to improved vision in
these infants.
Cryotherapy emerged as the standard treatment for
acute phase ROP in the 1980’s, following publication of the results of
Cryotherapy for Retinopathy of Prematurity Cooperative Group (CRYO
ROPCG) trial [4]. The study showed significant decline in the
progression of threshold ROP at which stage the risk of blindness if
untreated was 50%. Cryotherapy also significantly reduced the
unfavorable structural outcome of threshold ROP to 49.3% at 3 months and
45.8% at 12 months [5]. Cryotherapy requires a general anaesthetic or
sedation and ventilation. Conjunctival dissection is needed in posterior
disease to enable access for the cryoprobe. Complications are
post-operative pain, lid edema, laceration and hemorrhage of
conjunctiva, preretinal and vitreous hemorrhage. Laser (light
amplification by stimulated emission of radiation) therapy evolved as
the primary modality of treatment in 1990’s, with lesser complications,
and provided blindness prevention as effectively as cryotherapy.
However, the total cost of required equipment for laser therapy was much
higher than for cryotherapy. This factor may have to be considered for
the developing countries of the world, where the incidence of ROP is on
the rise. The complications reported with laser are burns of the cornea,
iris and lens, hyphema, retinal hemorrhages and choroidal rupture [6].
Hence, the need for newer modalities of treatment, which require less
skill and equipment. Some of these novel modalities are discussed here.
Vascular Endothelial Growth Factor (VEGF)
Two theories exist on the pathogenesis of ROP. The
mesenchymal spindle cells, exposed to hyperoxic extra uterine
conditions, develop gap junctions. These gap junctions interfere with
the normal vascular formation, triggering a neovascular response, as
reported by Kretzer and Hittner [7].
Ashton theorized that 2 phases exist [8]. The first phase
is at the time of premature birth, the infant retina becomes hyperoxic
(even in room air) with decreased levels of VEGF. For a period of time,
vessel formation is halted at the interface between the vascular and
avascular retina (Phase I, clinically 22–30 weeks postmenstrual age). As
the eye grows, the avascular retina continues to increase in size
without accompanying inner retinal vessels. This creates a peripheral
area of hypoxic retina, resulting in increased levels of VEGF, which
stimulates angiogenesis (pathological neovasculari-sation) at the
interface between the vascular and avascular retina (Phase II,
clinically 31-45 weeks postmenstrual age). This two phase process leads
to vision-threatening ROP most frequently in extremely immature infants
with other comorbidities of prematurity (risk factors for ROP). These
new vascular channels are not mature and do not respond to proper
regulation. Although many causative factors, such as low birth weight,
prematurity, and supplemental oxygen therapy are associated with ROP,
several indirect lines of evidence suggest the role of a genetic
component in the pathogenesis of ROP [9]. Genetic polymorphism may alter
the function of the genes that normally control retinal vascularization,
such as VEGF, which may also be involved in the pathogenesis of ROP.
Anti-Vascular Endothelial Growth factor Therapy
Vascular endothelial growth factor A (VEGF-A) is a
known promoter of angiogenesis and is upregulated by hypoxia. It
interacts with endothelial cells via its two membrane-bound receptors,
VEGFR-1 and VEGFR-2, which belong to the tyrosine kinase receptor family
[10]. VEGFR-1 controls the assembly of tubes and functional vessels by
endothelial cells. VEGFR-2 promotes the differentiation and
proliferation of endothelial cells. Its expression is increased by
hypoxia and potentiated by VEGF [11,12]. An association with
ischemia-induced proliferative diseases has clearly been established
[13]. VEGF-A plasma levels of infants born at term were reported to vary
from 200 to 450 pg/mL during the first few weeks of life and decline
rapidly to adult range levels of 10-110 pg/mL within a few months after
birth [14]. Interestingly, plasma VEGF-A levels in preterm infants were
found to be relatively low and stable during the first 7 days of life
(48 pg/mL, SD 6) [15]. Similarly, in a mouse model of ischemia induced
retinal revascularisation, immunohistochemistry for VEGFR-1 and VEGFR-2
revealed that the immunoreactivity of VEGFR-2 was increased in the
vessels near the avascular area, whereas the pattern of VEGFR-1
expression in the hypoxic retina was almost the same as that of control
animals [16]. VEGFR-2 expression was found to be mainly associated with
pathological neovascularisation and less with physiological postnatal
vessel development [17]. One study found VEGF-A to be elevated in the
subretinal fluid in ROP stage 4 (mean 44.16 ng/mL, SD 18.72) and greatly
reduced in stage 5 [18]. In contrast, another study investigated 38
cases of stage 5 ROP at the time of vitrectomy and found increased VEGF
immunoreactivity in the vascularised regions of fibrovascular membranes
[19].Thus the role of vascular endothelial growth factor (VEGF) in
neovascularization and vascular permeability of ROP is established [20].
Hence, therapy for ROP is directed at treating the underlying
pathogenesis by decreasing VEGF levels (specifically VEGF-A), either by
completely ablating the peripheral avascular retina that produces the
VEGF (LASER therapy) or by inactivating VEGF by binding it after its
production (anti-VEGF therapy).
Bevacizumab, a humanized recombinant antibody, that
inhibits the biological activity of VEGF, has been widely used as a
off-label treatment for ocular angiogenesis disorders, including
age-related macular degeneration [21], proliferative diabetic
retinopathy (PDR) [22] and neovascular glaucoma [23]. It is a complete
antibody rather than an antibody fragment like ranibizumab.
The BEAT–ROP study (Bevacizumab eliminates the
angiogenic threat of ROP) is a Phase II study (intravitreal bevacizumab
injections versus conventional LASER surgery for ROP). In one study,
after injection of bevacizumab as the initial treatment, reduced
neovascular activity was seen on fluorescein angiography in 14 of 15
eyes under study. In three eyes, a tractional retinal detachment
developed or progressed after bevacizumab injection. No other ocular or
systemic adverse effects were identified [24]. Bevacizumab has been
shown in a small case series to temporarily slow vasculogenesis and
permanently halt angiogenesis, usually with a single intravitreal
injection, when used for vision-threatening ROP stage 3 (acute phase ROP
including AP–ROP) [25].
Bevacizumab, given to extremely immature infants by
intravitreous injection as low dose monotherapy (0.625 mg in 0.025 mL of
solution) or upto 0.75 mg, has not shown systemic or local toxicity. The
most likely local complications of the injections are infectious and
traumatic. Infections can be avoided by strict sterile technique
followed by one week of appropriate antibiotic ophthalmic drops. Trauma
may occur to the lens because the injection is given too anteriorly. No
systemic complications have been encountered to date whether bevacizumab
given alone, or in combination with LASER surgery (when the retinal
barrier has been breached).
Thus, bevacizumab alone for ROP stage 3 may become
not just an adjunct to laser therapy or vitrectomy, but primary
treatment replacing laser therapy as standard of care, if efficacy and
safety are validated by evidence-based data. Laser eventually may be
contraindicated because it is a very destructive therapy that is never
completely without residual effects and targets the same pathogenic
substance: VEGF. ROP stages 4 and 5 will continue to occur due to late
diagnosis of acute disease from less than adequately screened nurseries
and in these desperate cases vitrectomy will be required, perhaps with
bevacizumab as an adjunctive therapy.
This novel drug therapy is being tried by many
neonatologists in developing countries where facilities for laser or
cryotherapy may not be available. Moreover, bevacizumab is a relatively
easy and inexpensive treatment. In developing countries like India,
where the per capita GDP is USD 543 (March 2006), the majority of
patients cannot afford to pay USD 730 for a single vial of bevacizumab
[26]. Therefore, efforts were made to make 20 fractions of 0.2 mL from a
single vial, thus decreasing the cost to USD 38 per injection [27].
Another Anti-VEGF drug researched is Pegaptanib
sodium. The initial experience using pegaptanib sodium to treat ROP
suggest that the medication is well tolerated, and helps to quieten the
vascular activity in eyes with severe posterior disease, but does not
prevent the development of retinal detachment. This thus is still under
study as an alternate anti VEGF therapy. We would advocate caution;
however, before introducing this new, potentially exciting therapy,
especially since large randomized clinical trials are still to be
conducted [28].
Insulin like Growth Factor – I
It was shown that lack of insulin-like growth factor
I (IGF-I) in knockout mice prevents normal retinal vascular growth,
despite the presence of vascular endothelial growth factor, important to
vessel development [29]. Results from studies in premature infants
suggest that if the IGF-I level is sufficient after birth, normal vessel
development occurs and retinopathy of prematurity does not develop [30].
When IGF-I is persistently low, vessels cease to grow, maturing
avascular retina becomes hypoxic and vascular endothelial growth factor
accumulates in the vitreous. As IGF-I increase to a critical level,
retinal neovasculari-zation is triggered [31]. These data indicate that
serum IGF-I levels in premature infants can predict which infants will
develop retinopathy of prematurity and further suggests that early
restoration of IGF-I in premature infants to normal levels could prevent
this disease [32]. A study from Sweden [33] studied the pharmacokinetics
and dosing of intravenous insulin-like growth factor-I and IGF-binding
protein-3 complex in preterm infants. The recombinant human IGF-I (rhIGF-I/rhIGFBP-3)
equimolar proportion was effective in increasing serum IGF-I levels and
administration under study conditions was safe and well tolerated.
IGFBP-3 is a protein that binds to IGF-I and regulates the availability
and activity of IGF-1. Mecasermin rinfabate mimics the effects of this
natural protein complex (IGF-1/IGFBP-3) in the bloodstream and is able
to stay in the body for a longer period. The drug is under study and no
studies to prove its efficacy have been published yet. The cost efficacy
of this drug is quite disappointing when compared to the other
modalities.
Granulocyte Colony–stimulating Factor
Granulocyte colony-stimulating factor (GCSF), a
biologic at commonly used to increase leukocyte counts in neutropenic
adults and children might also have a regulatory effect on
vasculogenesis and thus prevent ROP. GCSF has been shown to increase
levels of insulin-like growth factor-1(IGF-1), which supports the
normal, measured, calm vascularisation of the retina. Conversely,
falling levels of IGF-1 appear to set off a disorderly, aggressive
vascularisation of the retina. In mouse oxygen-induced retinopathy
model, G-CSF significantly reduced vascular obliteration (P
<0.01) and neovascular tuft formation (P <0.01). G-CSF treatment
also clearly rescued the functional and morphologic deterioration of the
neural retina [34]. So GCSF, which is given to many premature infants,
might be used to prevent ROP, particularly in infants with falling IGF-1
levels. The beauty of this approach, as opposed to, say, using
bevacizumab, is that it doesn’t destroy VEGF. This hunch led to a
retrospective chart review of 213 infants who, for non ophthalmic
reasons, received GCSF in the neonatal intensive care unit at the
University of Louisville. Fifty infants with low birth weight and a
gestation of 32 weeks and under were matched to a control group that did
not receive GCSF. Only 10% of the infants who received GCSF required
laser treatment, compared with 18.6% of the controls. Further, those
babies in the GCSF group who required laser had an exceptionally low
average birth weight and had received relatively low doses of GCSF [35].
Filgrastim is a human granulocyte colony-stimulating
factor (G-CSF)‚ produced by recombinant DNA technology. It’s potential
role in the prevention of ROP is now being studied, hence the dose
required and side effects are still not documented. As this drug is more
easily available in India with costs ranging from 2500 – 3000 INR for a
300 mcg/mL vial, this would be a beneficial adjunct to the treatment of
ROP in a developing country like ours.
Jun Kinases (JNK) Inhibitors
The Jun kinases (JNK) belong to the mitogen-activated
protein kinase (MAPK) family [36]. These kinases, which are encoded by
three separate loci, Jnk1-3, regulate key cellular
processes such as cell proliferation, migration, survival, and cytokine
production. In cell culture studies, a nonspecific JNK inhibitor was
shown to affect VEGF mRNA stabilization [37]. Hence, JNK1 is a critical
factor in hypoxia induced retinal VEGF production and that it promotes
hypoxia induced pathological angiogenesis. JNK1 deficiency or JNK
inhibition results in reduced pathological angiogenesis and lower levels
of retinal VEGF in an experimental model of ROP [38]. Intravitreal
injection of a specific JNK inhibitor decreases retinal VEGF expression
and reduces pathological retinal neovascularization without obvious side
effects. These results strongly suggest that JNK1 plays a key role in
retinal neoangiogenesis and that it represents a new pharmacological
target for treatment of diseases where excessive neoangiogenesis is the
underlying pathology [39]. D-JNKi – the specific JNK-1 inhibitor is the
drug that is under study for the prevention of ROP.
Gene Therapy
A series of small studies have investigated the
association of gene and severe ROP or failure of treatment [40]. They
have implicated mutations and polymorphisms in the Norrie disease
pseudoglioma (NDP) gene, Endothelial nitric oxide
synthetase (eNOS) gene and Vascular endothelial growth
factor (VEGF) gene. Unfortunately, most studies do not show a
significant association of genetic abnormalities and ROP. The influence
of genes on the occurrence, progression and severity of ROP warrants
further investigation in various populations and in larger cohorts [41].
The answer may lie in manipulating transcription factors and alternative
splicing of putative genes involved in ocular NV, tipping the
microenvironment to an anti-angiogenic state [42]. We believe that
through techniques in gene therapy, alternative splicing and RNA
interference, we may meet with greater success in restoring ocular ‘angiogenic
privilege’. Chowers [43] gave his view of the future by demonstrating
that gene transfer into blood vessels is possible in a rat model of
retinopathy of prematurity. In their studies, Chowers tested retrovirus,
adenovirus and herpes virus based vectors, shuttling a
galactosidase reporter gene for expression in retinal blood
vessels in rodents undergoing oxygen induced retinal
neovascularisation. Interestingly, they found that adenovirus
offered the best efficiency in expression in retinal blood
vessels compared with all the other vectors. Moreover, the
adenovirus expression was specific to the blood vessels of the
inner retina and did not appear to be expressed in the deeper
neural retina. Hence, gene therapy has a definite future in this
blinding eye disease.
Funding: None; Competing interests:
None stated.
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