|Disruption of Bevacizumab (Avastin) Activity by Vitreous Matrix Gel
David Ta-Li Liu1*, Li Xu2, Chi-Pui Pang2, Dennis Shun-Chiu Lam2 and Gary Hin-Fai Yam2
|1Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, China
|2Prince of Wales Hospital, Hong Kong, China
||Dr. David Ta-Li Liu
Department of Ophthalmology & Visual
Prince of Wales Hospital
Hong Kong, China
|Received November 24, 2010; Accepted February 21, 2011; Published February
|Citation: Liu DT, Xu L, Pang C, Lam DS, Yam GH (2011) Disruption of
Bevacizumab (Avastin) Activity by Vitreous Matrix Gel. J Clinic Experiment
Ophthalmol 2:140. doi:10.4172/2155-9570.1000140
|Copyright: © 2011 Liu DT, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.
|Purpose: To investigate the modification effect of ocular vitreous matrix gel on bevacizumab activity.
|Methods: Bevacizumab was pre-incubated in native or denatured rabbit vitreous mixture prior to addition to
human umbilical vein endothelial HUVEC-2 cells in an in vitro scratch-wound assay. The effect of wound closure was
monitored by cell density in denuded area. Intracellular VEGF signaling was evaluated by western blotting for pan-Akt
and phosphorylated Akt (at Ser-473). Results by both methods were evaluated and compared.
|Results: Presence of bevacizumab (312 µg/ml) attenuated the stimulating effect of VEGF (10 ng/ml) on HUVEC
proliferation and migration. However, the inhibitory effect of bevacizumab on VEGF was markedly curtailed if
bevacizumab was pre-incubated in rabbit vitreous for 7 days before treatment. This interesting de novo inhibitory effect
of vitreous on bevacizumab was not observed when bevacizumab was pre-incubated in thermally denatured vitreous.
Western blotting showed bevacizumab blocked Akt phosphorylation but this inhibitory activity was again reduced if
bevacizumab had been pre-incubated with native vitreous.
|Conclusions: Our results demonstrated for the first time the inhibitory proteomic modification or disruption
of bevacizumab molecule by native vitreous protein matrix. The effects were probably enzymatic modifications of
bevacizumab molecules in vitreous, rather than simple diffusional or imbibitional loss of bevacizumab into the retina.
The implication of this vitreal modification of bevacizumab molecule should not be underestimated as it may carry
important clinical bearing on the actual pharmacodynamic therapeutic effect of bevacizumab at choroidal vascular
|Angiogenesis, a process of vessel sprouting from pre-existing
vasculature, is crucial in various biological processes, including
embryonic vascular development, tissue differentiation, organ
development and wound healing. In pathological states, angiogenesis
is associated with tumorigenesis, psoriasis, chronic inflammatory
disorders and ocular neovascularization [1,2]. In human eye, it is a
common cause of severe visual impairment or even blinding diseases
with relentless deterioration. Among them, corneal neovascularization
caused by infection, chemical burns or contact lens-induced keratopathy
obliterates the transparency of cornea . Iris neovascularization
restricts the anterior chamber angle and may lead to neovascular
glaucoma . Retinal neovascularization is often associated with the
proliferative diabetic retinopathy and retinopathy of prematurity
. And lastly, choroidal neovascularization in wet type age-related
macular degeneration is prone to leakage and fibrous proliferation,
which could trigger off cascades of complication like retinal edema,
retinal or vitreous hemorrhage and tractional retinal detachment .
|Inhibitors to vascular endothelial growth factor (VEGF) and
its receptor signaling represent an effective treatment for these
dreadful proliferative vascular diseases. Bevacizumab (Avastin), a
VEGF-neutralizing antibody approved by The US Food and Drug
Administration, is a potent chemotherapy agent for cancers, like lung,
colon and breast . Its off-labeled use has been shown to reduce ocular
neovascularization [8-10]. Intravitreal bevacizumab has been reckoned
as the most direct, clinically most effective and easy-to-use drug
delivery modality to treat retinal and choroidal neovascularization.
However, the detailed pharmacokinetics and pharmacodynamics of
the intravitreal fate of bevacizumab molecules are largely lacking. In
the intravitreal delivery of bevacizumab, vitreous matrix fibrils are
the natural conduit for the intraocular passage of the bevacizumab
molecules to come into contact with the target tissues like retinal or choroidal vasculature. Within the finite period of time of transport
of the bevacizumab molecules across this collagenous matrix bed,
it is conceivable that this complex proteinacious scaffold may exert
irreversible physiochemical as well as biochemical modifications or
interactions on the bevacizumab molecules. Nevertheless, very little has
been known of these very important in vivo molecular modifications,
which may be akin to the well known "first-pass effect" of the
enterohepatic circulation. It is still an enigma about the interaction of
intravitreal bevacizumab molecules and their bathing vitreous proteinrich
matrix. This is an important area to explore as this may skew the
overall intravitreal activity and efficacy of bevacizumab molecule in
inhibiting choroidal/retinal angiogenesis.
|In this study, we observed that bevacizumab activity was modified
by rabbit vitreous in vitro, irrespective of species specificity. Though
there was no effect on endothelial cell growth and viability, rabbit
vitreous downgraded the inhibitory action of bevacizumab on VEGF
signaling, as shown by the reduced phosphorylated Akt ratio, and
migration of human umbilical vein endothelial cells in an in vitro scratch wound assay.
|Rabbit vitreous and bevacizumab treatment
|Twenty-four New Zealand albino rabbits with age of 6 months
old and no detectable ocular defects were used. Vitreous humor was
withdrawn from pars plana position with a 3 ml syringe and needle
25G x5/8 under sterilized condition. Collected vitreous sample was
centrifuged at 1,400 g at 4°C for 30 minutes to remove cellular debris.
Bevacizumab (25 mg/ml, Roche, Basel, Switzerland) was mixed with
vitreous (native or denatured at 95°C for 5 minutes) at a ratio of 3.12 µg
bevacizumab per ml vitreous. The mixture was incubated at 37°C for 7
days (about 2 half-lives of bevacizumab in vivo)  and immediately
used for treatment.
|Endothelial cell Culture and migration assay
|Immortalized primary human umbilical vein endothelial cell line
HUVEC-2 (BD Biosciences, Becton Drive, New Jersy) was cultured in
Endothelial Cell Growth medium (PromoCell, Heidelberg, Germany)
added with SupplementMix from the manufacturer on 0.1% gelatincoated
surface. HUVEC-2 within the first 8th passages was used for a
scratch wound cell migration model (Figure 1K). In brief, HUVEC-2
grown to confluence on gelatin-coated surface were starved in medium
with 0.2% FBS (Invitrogen, Carlsbad, CA) for 4 hours. A scraping tool
(1 mm by width) removed a strip of cell monolayer to provide a margin
of denuded area. The dislodged cells were immediately removed and
stationary cells were treated with bevacizumab/vitreous-supplemented
medium with or without 10ng/ml VEGF. The number of cells in denuded
area was monitored at 0, 24 and 48 hours by phase-contrast microscopy
using a 5x objective. With Photoshop CS3, images of the same area were
aligned and cells were quantified and expressed as number of cells per
mm2 area. Six images were analyzed for each treatment and the mean
cell density was calculated, analyzed by paired Student's t-test.
|Cells were lysed in ice-cold RIPA (radioimmunoprecipitation
assay) buffer freshly added with PhosSTOP (Roche), protease inhibitor cocktail (Roche) and 1 mM phenylmethyl sulfonylfluoride (Sigma, St
Louis, MI) and soluble protein was analyzed by western blotting with
rabbit polyclonal antibodies against human phospho-Akt (Ser-473, Cell
Signaling, Danvers, MA), pan-Akt (Cell Signaling) and glyceraldehyde
3-phosphate dehydrogenase (GAPDH, Sigma), respectively, followed
by enhanced chemiluminescence. Band intensity was analyzed by
Quantity One Imaging software (BioRad, Hercules, CA). The active Akt
was calculated as the ratio of phospho-Akt to pan-Akt and compared
among samples, analyzed by paired Student's t-test.
|VEGF inhibition by bevacizumab was not affected by 37°C
|We examined the effect of bevacizumab on VEGF activity by an in
vitro HUVEC scratch-wound assay. Our analysis showed that VEGF (10
ng/ml) induced wound closure by HUVEC growth and migration in the
scratch wound area. This effect was evident at 24 hours after treatment
(with VEGF: 25.0±4.4 cells/mm2 wound area; without VEGF: 14.3±3.4
cells/mm2 wound area, P=0.008, paired Student's t-test) (Figures.
1B, 2A). Simultaneous treatment with bevacizumab (312 µg/ml) and
VEGF resulted in significantly fewer cells in the wound area (12.6±3
cells/mm2), when compared to VEGF-only (25.0±4.4 cells/mm2 wound
area) (P=0.0033, paired Student's t-test) (Figure 1C and Figure 2A). We
detected similar reduction of HUVEC number when bevacizumab was
pre-incubated at 37°C for 7 days before treatment (14.0±4.2 cells/mm2
wound area) (Figure 1D and Figure 2A). Treatment with 10 µM PP2 to
block src kinase signaling in downstream VEGF cascade resulted in a
drastic disappearance of cells (4.4±1.9 cells/mm2 wound area).
|Vitreous down-regulated bevacizumab activity
|Bevacizumab was incubated in cell-free rabbit vitreous (native or
heat-denatured) at a concentration of 3.12 g/ml vitreous for 7 days. At
time of cell treatment, 1 volume of bevacizumab/vitreous was added to
9 volumes of medium with 0.2% FBS to attain a working concentration
of 312 µg/ml bevacizumab in medium. We detected that rabbit vitreous
modified the action of bevacizumab on VEGF inhibition. The VEGF
activity was maintained when bevacizumab was pre-incubated with
native vitreous and this resulted in a significant increase of HUVECs
in the wound area (21.5±4.8 cells/mm2), when compared to treatment
without native vitreous (12.5±3 cells/mm2 wound area) (P=0.0223,
paired Student's t-test) (Figure 1E and Figure 2A). However, the VEGF
suppression by bevacizumab was not altered when bevacizumab
was pre-incubated with denatured vitreous, and we observed fewer
HUVECs present in the wound area (14±2.1cells/mm2).
|We also examined the effect of rabbit vitreous on HUVEC growth and migration in our wound assay. In the presence of VEGF (10 ng/ml),
incubation with either native or denatured vitreous did not significantly
affect the number of HUVECs present in the wound area, though a
minor reduction of cells was observed for the denatured vitreous (Figure
1G and Figure 1H). The cell density was 23.3±5.2 cells/mm2 for native
vitreous and 19.1±5.3 cells/mm2 for denatured vitreous, respectively
(Figure 2B). Without VEGF supplementation in culture, there was a
drop in HUVEC number in the wound area after the incubation with
denatured vitreous (12.2±2.5 cells/mm2). The decrease was significant
when compared to that with VEGF (P=0.005 paired Student's t-test)
(Figure 2B). No difference was found between cells with native vitreous
with or without VEGF (Figure 2B).
|Vitreous modified Avastin effect on VEGF/Akt signaling
|Soluble cell lysate was obtained at 4 and 24 hours for protein analysis
of Akt activation downstream of VEGF and its receptor interaction.
The specific signals of both phospho-Akt (Ser-473) and pan-Akt were
measured by band densitometry and Akt activation was represented
by the ratio of phospho-Akt to pan-Akt. All samples were compared
to untreated control cells. We observed Akt activation at 4 hours
after various treatments, however the effect was subsided at 24-hour
(Figure 3A and Figure 3B). Addition of VEGF (10 ng/ml) substantially activated Akt through the phosphorylation at Ser-473 (sample 2).
It was about 3 fold higher than that of untreated control (sample 1)
(Figure 3C). Similar Akt activation was found in samples treated with
VEGF and rabbit vitreous (native or denatured) (samples 3 and 4). The
treatment of bevacizumab at 312 µg/ml alone (sample 8) or in denatured
vitreous (sample 6) suppressed Akt activation caused by VEGF and the
reduction was significant when compared to VEGF-only cells (sample
3) (P<0.005, paired Student's t-test). However, when bevacizumab was
pre-incubated with native vitreous, the VEGF-induced Akt activation
level was maintained (sample 5), indicating a reduction of bevacizumab
activity on VEGF suppression.
||Figure 1: Vitreous down-regulated bevacizumab activity. An in vitro HUVEC
scratch-wound assay was performed to examine the effect of vitreous on
bevacizumab activity on VEGF. (A-J) Representative phase contrast images to
show the proliferation and migration of HUVEC in the denuded area produced
by pipette tip scrapping. (A) Untreated control; (B) cells treated with VEGF (10
ng/ml); (C) VEGF and bevacizumab (312 µg/ml); (D) VEGF and bevacizumab
pre-incubated at 37°C for 7 days; (E) VEGF and bevacizumab pre-incubated in
native vitreous at 37°C for 7 days; (F) VEGF and bevacizumab pre-incubated
in denatured vitreous at 37°C for 7 days (G) VEGF and native vitreous; (H)
VEGF and denatured vitreous; (I) native vitreous and (J) denatured vitreous.
(K) A schematic diagram showing the six images captured in the scratch-wound
assay. VEGF, vascular endothelial growth factor.
||Figure 2: (A and B) Histograms showing the number of cells per unit of denuded
area in various treatments. *P<0.05 and **P<0.01 by paired Student's t-test.
HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial
growth factor; N, native vitreous; DN, denatured vitreous.
||Figure 3: Western blotting analysis of Akt activation in RF/6A cells after
various treatments. (A) 4-hour treatment; (B) 24-hour treatment and (C)
histogram showing the ratio of Akt phosphorylation normalized with GAPDH.
Transient up-regulation of phosphorylated Akt in total Akt was resulted from
VEGF (10 ng/ml) treatment. Addition of bevacizumab (312 µg/ml) blocked the
Akt activation by VEGF but this effect was reduced when bevacizumab was
pre-incubated with rabbit vitreous prior to addition to cells. Bevacizumab activity
to block Akt phosphorylation was maintained in denatured vitreous. *indicates
P<0.05, paired Student's t-test. VEGF, vascular endothelial growth factor; N,
native vitreous; DN, denatured vitreous; GAPDH, glyceraldehyde 3-phosphate
|Bevacizumab (Avastin), a recombinant full-length humanized
monoclonal antibody, specifically binds to the soluble biologically active
VEGF isoforms. It prevents receptor binding of VEGF and inhibits
endothelial cell proliferation and vessel formation . In recent years,
bevacizumab has been widely adopted to treat not only choroidal
neovascularization associated with AMD but also neovascularization
in myopia, uveitis, iris and corneal neovascularization [13,14].
Pharmacokinetic studies have shown that intravitreal injection is the
most effective route of administration for intraocular tissue . The
vitreal bevacizumab concentration declined mono-exponentially
with a half-life of 4.32 days and about 0.8% of injected bevacizumab
was detected in vitreous after 30 days.11 Minute amount (in range of
nanogram) of bevacizumab was detectable in aqueous and vitreous
humor of fellow eye and in the plasma [16,17]. However, no report on
the activity of bevacizumab in vitreous is available, yet most studies
had emphasized the beneficial outcome of bevacizumab in alleviating
neovascularization development [9,10,18-22].
|In this study, we recruited a rabbit vitreous model to study the
proteolytic activity of vitreous substance on bevacizumab molecules. In
our in vitro scratch-wound assay, the inhibitory effect of bevacizumab
on VEGF-induced HUVEC growth and migration was reduced after
the pre-incubation with native vitreous at 37°C for 7 days (~
of bevacizumab in vivo). This was further substantiated by the preserved
Akt activation (phosphorylation at Ser-473 site), which acts as the
intracellular signaling in VEGF cascade. This indicated that no change
of bevacizumab due to its self-modification or degradation occurs at
37°C for 7 days and bevacizumab was effective in prohibiting HUVEC
proliferation and migration in the denuded area driven by VEGF.
However, bevacizumab activity was altered or modified by vitreous,
in which the high content of serum proteins, proteases or metabolic
enzymes might modulate or degrade bevacizumab molecules [23,24].
Serine proteases, such as plasmin, can degrade fibrin, fibronectin and
proteoglycan core proteins, and similar susceptible sites could be present
in the bevacizumab structure. In addition, matrix metalloproteinases
(MMPs) and cysteine proteases are involved in extracellular matrix
remodeling. Gelatinase A (or MMP-2) can denature collagen in
vitreous or surrounding tissues, supporting its possible role in vitreous
liquefaction in aging eyes . Our study further revealed that the
vitreous effect on bevacizumab activity could be complex and fraction
of bevacizumab remained active after vitreous incubation. This could
be due to the possible co-existence of other vitreous proteins that might
help preserving the integrity and functional activity of bevacizumab.
Previously, it had been reported the plasma protein in vitreous could
function to protect ocular tissue against oxidative stress .
|The reduced bevacizumab activity by vitreous might be associated
with the breakdown of bevacizumab or modification of bevacizumab
structure. In our recent study using high performance liquid chromatography method to quantify the vitreal bevacizumab levels after
intravitreal injection to rabbit eyes, we observed that not only the level
decreased by time after injection but also the change of structure in situ (manuscript submitted). The modifications were mainly in the heavy
chain, both the variable and constant regions, although minor changes
were also detectable in the light chain. This interesting observation
was substantiated by results from peptide mapping and tandem mass
spectrometry analyses. Hence, we hypothesized that bevacizumab
structure and activity could be altered and modified by vitreous and
this change was not species specific.
|In vitro studies have indicated that vitreous modulates retinal
cell survival and proliferation [27,28]. A porcine vitreous fraction
of molecular mass smaller than 1000 Da promoted the growth of rat
retinal precursor cells and this effect was augmented by albumin, a
major protein in the vitreous fluid . Similar phenomenon occurred
in the porcine aqueous humor on rat retinal precursor cells. Ascorbic
acid could be a candidate factor in humor contributing to such changes.
When applied to primary human retinal pigment epithelial (RPE) cells,
vitreous modified gene expression including down-regulation of genes
associated with RPE differentiation, and up-regulation of genes in focal
adhesion, stress and inflammation (transforming growth factor-β /
bone morphogenic protein-2 pathways) [29-31]. This suggested that
vitreous might promote the migratory potential of RPE cells, which
were transformed to more fibroblast-like morphology. This might be
the underlying pathogenesis of the proliferative vitreoretinopathy in
which RPE cells dissociate from the Bruch's membrane and proliferate
in the vitreous. The cells, with a more fibroblast-like appearance, are
detected in the epiretinal membranes and may involve in membrane
contraction leading to traction retinal detachment.
|In addition, vitreous contains mitogens to stimulate cell
proliferation and gene regulation associated with inflammation, cell
cycling, intracellular transduction and growth . In this study, we
demonstrated that native vitreous might promote endothelial cell
growth. This simulation is due to the cytokine, chemokine or plasma
activity in the humor fluid. These heat labile factors together with
hyaluronic acid (HA), glycosminoglycans or chondroitin sulfate play
pivotal roles in cell survival and homeostasis. HA is a major nonprotein
glycosminoglycan component of ECM and is richly present
in the vitreous. Its principle ligand is the cell surface glycoprotein
CD44, which is present in both retina and choroids . Association
between HA and CD44 has been implicated in many physiological
processes involving cell-cell or cell-ECM interactions . Its effect
on cell survival and proliferation might involve the down-regulation
of p27kip1 cyclin-dependent kinase inhibitor and activation of cell
growth protein (such as retinoblastoma protein) .
|In conclusion, we observed the modulation of bevacizumab activity
by vitreous humor. Our result provided the first evidence to show the
possible metabolic fate of the bevacizumab molecules in the usually
neglected passage through the vitreous matrix fibrils, well before their
hitting on the targeted retinal tissue layers or choroidal vasculatures,
setting-off a cascade of physiochemical interactions and promulgating
the beneficial effect of bevacizumab in retinal and choroidal
neovascularizations. The implication of this interesting post intraocular
injection vitreal modification of bevacizumab molecule should not be
underestimated as it might carry important clinical bearing on the
actual pharmacodynamic therapeutic effect of bevacizumab at retinal
as well as choroidal vascularture level. This warrants further clinicalpathological
study as vitreous modification might be ubiquitous in the
arena of intravitreal pharmacology.
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