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From the Department of Microbiology,*
University of
Tennessee, Knoxville, Tennessee; and the Departments of Pediatrics and
Surgery,
Children’s Hospital of Los Angeles,
University of Southern California, Los Angeles, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present report, we have tested this idea choosing for study a
cytokine endothelial monocyte-activating polypeptide II (EMAP II) shown
previously to diminish neovascularization in a tumor system as well as
in corneas implanted with the angiogenic factor basic fibroblast growth
factor.10
The potential advantage of EMAP II is that this
cytokine seems to inhibit growing blood-vessel endothelial
cells.10-12
Thus EMAP II
might act against a range of angiogenesis factors such as could be
induced by HSV infection. Several effects of EMAP II on neovessels have
been described. These include direct apoptosis of vascular endothelial
cells by either activating caspase 310
or Fas-associated
death domain and down-regulation of Bcl-211
as well as
indirect effects on these cells by up-regulated tumor necrosis factor
receptors so making the endothelial cells subject to tumor necrosis
factor-
-induced apoptosis.13,14
EMAP II may also damage
vascular beds by causing up-regulation of tissue factor that in turn
leads to local activation of clotting factors and hence small vessel
occlusion.15
Besides effects on vascular endothelial
cells, EMAP II has potential unwanted side effects because in some
circumstances local or systemic pro-inflammatory effects have been
described.15,16
These effects seem to be fleeting and
considered as insignificant by some investigators.10
Thus,
on balance EMAP II represents an attractive candidate anti-angiogenesis
factor to test for inhibitory effects against herpetic ocular lesions.
Our results demonstrate that daily systemic and topical EMAP II administration to mice with HSV-infected eyes, led to diminished HSK lesions. The outcome seemed to result from the effect of EMAP II on new blood vessel development rather than from some direct or indirect antiviral activity. EMAPII was also shown to significantly reduce angiogenesis in the cornea caused by the potent angiogenic factor vascular endothelial growth factor (VEGF). This later effect seemed to result from the induction of apoptosis in developing blood vessel endothelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice (4 to 5 weeks old) obtained from Harlan Sprague-Dawley (Indianapolis, IN) were acclimated for 1 week before use in a specific pathogen-free animal colony accredited by the American Association for Accreditation of Laboratory Animal Care. All experimental procedures were conducted according to the Association for Research in Vision and Ophthalmology resolution on the use and care of laboratory animals.
Virus, Virus Corneal Infection, and Treatment Protocol
Wild-type RE HSV-1 was propagated and assayed on vero cells by a plaque assay. Mice were deeply anesthetized with methoxyflurane (Metofane; Pittman-Moore, Mondelein, IL). The corneal surfaces were scarified with a 27-gauge needle and 5 x 105 pfu of RE virus were applied in a 5-µl volume and gently massaged into the eyes. One hour before virus infection, the mice were given rEMAP II,10 in the presence of mouse serum albumin at a concentration of 1 mg/ml (Sigma Chemical Co., St. Louis, MO), 1 µg intraperitoneally and 1 µg topically per animal. The treatment was continued for 20 days. Separate studies were conducted in which EMAP II was not applied until day 7 after infection, and treatment was continued for 2 weeks. The level of lipopolysaccharide in the recombinant EMAP II was <15pg/ml as measured with a limulus amebocyte lysate (LAL) kit (Biowhittaker QCL-1000; Biowhittaker, Walkersville, MD). Signs of toxicity of EMAP II were looked for after systemic and local administration. Systemic signs would include failure to eat or drink, depression, and ruffed fur. Local effects in the eye would include ocular secretion and irritation, corneal edema, and cloudiness.
Eye Swab Viral Titration
At different time points after virus infection, eye swabs (four eyes at each time point) were taken using sterile swabs soaked in McCoy medium containing 100 UI/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Grand Island, NY). The swabs were then placed in tubes containing 500 µl McCoy medium and stored at -80°C. To detect HSV in swabs, the samples were thawed and vortexed, and 100 µl of each sample from individually marked mice was used for quantification of virus through recovery by standard PFU assay on vero cell cultures as described elsewhere.4
Clinical Scoring System
Mice were examined at different times after infection for the
development of clinical lesions by slit-lamp biomicroscopy (Kowa Co.,
Nagoya, Japan), and the severity of stromal keratitis and angiogenesis
were recorded as described elsewhere.4,17
The severity of
the HSK lesions and angiogenesis were also recorded by a
stereomicroscope (Leica, Germany) and image system (Hamamatsu, Japan).
Briefly, the clinical lesion score of HSK was described as 0, normal
cornea; 1, mild haze; 2, moderate haze, iris visible; 3, severe haze,
iris not visible; 4, severe haze and corneal ulcer; 5, corneal rupture.
In reference to the angiogenic scoring system, the method relied on
quantifying the degree of neovessel formation based on two primary
parameters: 1) the circumferential extent of neovessels (as the
angiogenic response is not uniformly circumferential in all cases); 2)
the centripetal growth of the longest vessels in each quadrant of the
circle; and 3) the longest neovessel in each quadrant was identified
and graded between 0 (no neovessel) and 4 (neovessel in the corneal
center) in increments of 
0.4 mm (radius of the cornea is 1.5 mm).
According to this system, a grade of 4 for a given quadrant of the
circle represents a centripetal growth of 1.5 mm toward the corneal
center. The score of the four quadrants of the eye were then summed to
derive the neovessel index (range, 0 to 16) for each eye at a given
time point. The extent of the neovessel ingrowth was also recorded by
direct measurement using calipers (Symbol of Quality, Biomedical
Research Instruments, Rockville, Maryland) under stereomicroscopy.
Corneal Lysate VEGF Enzyme-Linked Immunosorbent Assay (ELISA)
Corneas at different time points were isolated and put into RPMI 1640 without serum and stored at -80°C. The corneas were homogenized in an ice bath using a tissue homogenizer (PRO Scientific Inc., Monroe, CT) for 1 minute. The corneal lysates were collected and assayed for VEGF by sandwich ELISA. The two mVEGF monoclonal antibodies used were anti-mouse VEGF antibody and biotinylated anti-mouse VEGF antibody (R & D systems, Inc., Minneapolis, MN) and mVEGF164 was used as a standard (R & D systems, Inc.).
Histopathological and Immunohistochemical Staining
At various times after infection, whole eyes were fixed in 10% buffered neutral formalin, embedded in paraffin, and tissue sections were stained with hematoxylin and eosin as described previously.4 Sections were observed for thickness of the cornea, the presence of inflammatory infiltrates, neovascularization, and corneal perforation. For immunohistochemistry, eyes were removed and snap-frozen in OCT compound (Miles, Elkhart, IN). Six-µm sections were cut, air-dried, and fixed in cold acetone for 5 minutes. The sections were then blocked with 3% bovine serum albumin and stained with biotinylated anti-pan-endothelial antigen (Pharmingen, San Diego, CA). Sections were then treated with horseradish peroxidase-conjugated streptavidin (1:1000) and 3,3'-diaminobenzidine (Vector, Burlingame, CA) and counterstained with hematoxylin.
Corneal Micropocket Assay
The murine corneal neovascularization model followed the general
protocol of Kenyon and colleagues.18
Pellets for insertion
into the cornea were made by combining rhVEGF (40 µg, R&D system) and
sulcralfate (10 mg, Bulch Meditec, Vaerlose, Denmark) and hydron
polymer in ethanol (120 mg/1 ml ethanol; Interferon Sciences,
Brunswick, NJ) and applying the mixture to a 15 x 15
mm2
piece of synthetic mesh (Sefar America, Inc.,
Kansas City, MO). The mixture was allowed to air-dry and fibers of the
mesh were pulled apart, yielding pellets containing 90 ng of VEGF.
Pellets containing VEGF were inserted into a corneal pocket created 1
mm from the limbus at the lateral canthus of the eye under
stereomicroscope. Mice were then treated with vehicle or EMAP II (2
µg/day, 1 µg intraperitoneally and 1 µg topically) for the
following 4 days. Then the eyes were evaluated for corneal
neovascularization according to that previously
described.10
Briefly the number of vessels originating
from the limbus was counted over the entire orbit, and the area of
angiogenesis was calculated according to the formula for an ellipse.
A = [(clock hours) x 0.4 x (vessel length in mm)
x 
]/2. Each clock hour is equal to 30° at the circumference.
Cell Culture and Cell Death Detection
Human endothelial cells were isolated from umbilical cord vein by collagenase treatment as described elsewhere19 and used at passage 1 to 4. Human umbilical vein endothelial cells (HUVECs) were plated on two-well chamber slides and allowed to rest overnight, after which the cells were starved for 6 hours in RPMI 1640 without serum. The medium was replaced with either 10 ng/ml rhVEGF (R & D Systems) or 10 ng/ml rhVEGF plus 0, 10, 100, and 1000 ng/ml EMAP II, respectively. Briefly, the cells were incubated at 37°C for 24 hours. HUVEC apoptosis and corneal cell (corneal sections) apoptosis from both EMAP II- and vehicle-treated groups were monitored by using the in situ cell death detection kit, alkaline phosphatase (AP) (Boehringer Mannheim). Briefly, the cells on the chamber slides or the corneal sections were air-dried and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) solution for 1 hour at room temperature. The samples were then permeabilized in 0.1% Triton X-100, 0.1% sodium citrate for 2 minutes on ice. The slides were then washed with PBS and 50 µl of terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridinetriphosphate [dUTP] nick end labeling (TUNEL) reaction mixture was added and incubated in a humidified chamber for 60 minutes at 37°C. The HUVEC samples were then analyzed under fluorescence microscope (Leica, Germany) at this stage and the images were taken by imaging system. The corneal sections were processed further for converter-AP and substrate nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche Diagnostics GmbH, Mannheim, Germany) and analyzed by light microscopy.
Myeloperoxidase Assay
Myeloperoxidase, a marker for neutrophils—mainly existed in polymorphonuclear and macrophage—was detected according to the method of Bradley and colleagues20 as previously described. Briefly, myeloperoxidase was extracted from the homogenized single cornea by suspending the tested corneas in 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co.) in 50 mmol/L potassium phosphate buffer, pH 6.0, before homogenization in an ice bath for 1 minute. The specimens were freeze-thawed three times, after which homogenization was repeated. Suspensions were then centrifuged at 10,000 x g for 15 minutes and the resulted supernatants were assayed. Myeloperoxidase was assayed spectrophotometrically: A total of 200 µl of serially diluted corneal lysate samples and standard (myeloperoxidase, Sigma Chemical Co.) were added in duplicate and twofold diluted in 0.5% hexadecyltrimethylammonium bromide and then mixed with 100 µl of 50 mmol/L phosphate buffer, pH 6.0 containing 0.167 mg/ml O-dianisidine dihydrochloride (Sigma Chemical Co.) and 0.0005% hydrogen peroxidase. The color reaction was measured by an ELISA reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA) at 460 nm. Quantification was performed with Spectramax ELISA reader software version 1.2.
Statistics
Statistical analysis was performed using Student’s t-test. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a well-defined model of ocular infection with HSV,
susceptible BALB/c mice developed a chronic immunoinflammatory response
in the corneal stroma termed herpetic stromal keratitis
(HSK).2,21
Evident clinically 8 days after infection,
the lesion progresses in severity to peak levels 15 to 21 days after
infection.4,7
Initially after infection, a vigorous
neutrophil response is evident histologically in the cornea but this
response declines by 4 to 5 days after infection, as does the presence
of demonstrable infectious virus in corneal swabs (Figure 1, A and B)
. Usually neither infectious
virus, nor viral antigen can be demonstrated at the time HSK lesions
become clinically overt (Figure 1C)
. The undamaged cornea is normally
avascular, but after HSV infection, new blood vessel development
invades from the corneal limbus (Figure 2A)
. The angiogenesis increases and at
the time of severe HSK may reach the central corneal region (Figure 2C)
. At 5 to 7 days after infection, when virus is no longer
detectable and inflammatory lesions are inapparent clinically,
angiogenesis up to 0.6 to 0.8 mm beyond the limbus is usually present
(Figure 2B
and Figure 1D
). The means by which HSV infection results in
angiogenesis has not been defined. However, as shown in Figure 3
, HSV infection results in induction of
the potent angiogenesis factor VEGF. In corneal lysates made with mouse
corneas at various times after infection and tested with a capture
ELISA assay, VEGF levels in infected eyes were significantly above
those present in trauma control eyes at 48 hours after infection and
beyond. Application of UV-inactivated HSV to eyes failed to induce VEGF
levels above those observed in the trauma controls (data not shown). No
significant differences in VEGF values were observed between EMAP II-
and vehicle-treated groups at days 2 and 4 after infection with the
values of 31.5 ± 5.0 and 35.2 ± 6.9 pg/ml in the
vehicle-treated and 34.6 ± 7.5 and 33.2 ± 7.6 pg/ml in EMAP
II-treated mice. On the contrary, significant differences in VEGF
values at days 10 and 15 after infection (clinical phase) was noted
between the two groups with values of 81.3 ± 8.5 and 87.8 ±
9.0 pg/ml in the vehicle-treated and 53.4 ± 7.4 and 50.2 ±
8.5 pg/ml in EMAP II-treated mice.
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Because angiogenesis may represent an essential step in the
pathogenesis of HSK, the effect of the angiogenesis-blocking cytokine
EMAP II on lesion severity was investigated. Initially, the effect of
systemic and topical administration of various doses of EMAP II was
tested in a corneal micropocket assay for anti-angiogenic effects
against an optimal dose of VEGF incorporated into slow release pellets.
VEGF was chosen for study because this molecule is a potent angiogenic
factor22,23
and was induced in the eye after HSV
infection. In this model, we established that a daily dose of EMAP II,
1 µg intraperitoneally and 1 µg topically for 4 days, inhibited
angiogenesis (Figure 4B)
by up to 65%
(Figure 4C)
when compared to vehicle-treated animals (Figure 4A)
. In
addition, EMAP II was shown to cause DNA fragmentation by the TUNEL
assay in cultured HUVECs stimulated with rhVEGF (Figure 5A)
. Evaluation of 10 random high-power
fields of cultured HUVECs on chamber slides, revealed that there was a
14.5-fold increase in the number of apoptotic cells/high-power fields
in 1000 ng/ml EMAP II-treated cells (29 ± 9.3) as compared to
control (2.1 ± 2.0) (P 
0.001). Whereas
10 ng/ml EMAP II treatment had no significant difference in the number
of apoptotic cells induced compared to the control cells
(P 
0.05). This indicated that despite the
presence of VEGF, EMAP II is a potent inducer of apoptosis.
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: day 6 after infection cornea is
a representative example). However TUNEL-positive cells were not
observed in the neovessels of vehicle-treated groups. Some corneal
epithelial cells were also positive (Figure 5B)
for TUNEL staining but
few if any corneal endothelial cells showed signs of DNA fragmentation. To measure the effects of EMAP II on the severity of lesions in HSK, groups of mice were ocularly infected with HSV and treated daily with either EMAP II (as described above) or vehicle alone. In infected mice the extent of neutrophil infiltration in corneas was measured by a myeloperoxidase assay. Infected plus vehicle and infected plus EMAP II 24-hour samples had values of 121.5 ± 15.3 and 97.5 ± 9.2 µU, respectively, and 48-hour samples had values of 198.6 ± 14.6 and 211.4 ± 13.0 µU, respectively. These differences were not significantly different (P > 0.05). Similarly, viral titers were measured daily in EMAP II-treated and vehicle-treated mice. In both groups, virus was present until days 5 to 6 after infection and titers at all time periods were approximately the same.
To test the possible pro-inflammatory effect of EMAP II, neutrophil infiltration was measured at 24 hours after infection by the myeloperoxidase assay in control mice (scratched corneas only) as well as in test mice treated with EMAP II. Values of 24.6 ± 2.3 and 34.4 ± 7.2 µU, respectively, were recorded (these values were not significantly different).
The effects of EMAP II on HSV-induced lesions, as well as angiogenesis,
is shown in Figures 6 and 7
. As is readily apparent, mice given
EMAP II had significantly diminished HSK lesions at all phases of
clinical disease (Figure 6, B and D)
in comparison to controls (Figure 6, A and C)
. Lesions in EMAP II-treated animal were approximately
twofold reduced in severity (Figure 6G)
, but the effect was quite
variable. Thus 1 out of 24 treated eyes still showed severe lesions
(score of 5) yet others had no apparent lesions 2.0 (Figure 6F)
. In
control animals, examined at the same time points, only 2 out 24 eyes
showed a lesion score 1.0 and the rest of them were 3.0 (Figure 6E)
. The average score was 2.0 ± 0.3 in the EMAP II-treated group
versus 3.9 ± 0.4 in the control group at day 16 after
infection (P < 0.05) (Figure 6G)
.
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, throughout a 20-day
observation period, the angiogenesis score derived from both the
circumferential extent of angiogenesis and the distance from the limbus
was markedly reduced in EMAP II-treated animals at all time points
observed (Figure 7G)
. At day 8 after infection the neovascularization
in EMAP II-treated eyes extended 0.3 ± 0.2 mm from the limbus
(Figure 6B)
, whereas in vehicle-treated eyes extensive neovessels were
evident (0.8 ± 0.2 mm from the limbus) (Figure 6A)
. After day 16
after infection, in the control group, abundant neovessels were
evident, some already reaching the central region of the cornea
(1.3 ± 0.4 mm from the limbus) (Figure 6C)
. In contrast, in the
EMAP II-treated group at 16 days after infection, the average
centripetal length of the neovessels was only 0.6 ± 0.3 mm from
the limbus (Figure 6D)
. As measured by an anti-angiogenesis scoring
method (see Materials and Methods), at 16 days after infection, the
treated animal had a value of 8.5 ± 1.3, compared to the control
value of 15.3 ± 1.5 (P < 0.05) (Figure 7G)
. Immunohistochemical staining for neovessels using pan-endothelial
antigen also was used to show the extent of the corneal neovessel
ingrowth into the stroma at different time points after HSV-1 infection
(Figure 7, A to F)
. By this analysis, the number of the neovessels
existing in the EMAP II-treated group at all time points was
significantly less than that of the vehicle-treated group
(P < 0.05). At days 3 and 16 after infection,
the number of positively stained neovessels was 1.6 ± 0.9 and
38 ± 5.0/corneal section, respectively, in EMAP II-treated group
versus 6.0 ± 1.9 and 80 ± 9.3/corneal section,
respectively, in the control group (P < 0.05).
Whereas EMAP II treatment from the onset of infection diminished both
angiogenesis and HSK, delaying treatment until day 10, had no
significant measurable effects either on the severity of subsequent HSK
or the extent of angiogenesis compared to the control vehicle-treated
animal (see footnote Figure 6
). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ocular infection by HSV is the most common infectious cause of
blindness in North America.25
Approximately 20% of HSV
infections result in HSK, a lesion that requires prolonged management
with anti-inflammatory drugs and oftentimes corneal
transplantation.26
The pathogenesis of HSK, as
studied in a mouse model, involves a complex of events with stromal
keratitis mainly the consequence of a CD4+
T-cell-orchestrated inflammatory process.2-4,21
Such
CD4+ T cells are present in corneal tissues from
8 days after infection, a time when replicating virus has usually
disappeared.8,26
At this stage, neovascularization of the
normally avascular cornea is prominent. We assume that this is a
necessary event for CD4+ T cells to gain access
to the central cornea, a concept supported by the present data. Thus
mice treated with EMAP II had markedly diminished HSK lesions. As well
documented elsewhere, EMAP II may express potent anti-angiogenesis
activity.10
Such effects were demonstrated against
pathological angiogenesis in tumor models10,11,24
as well
as against physiological angiogenesis occurring during organ
development.12,27,28
EMAP II may also be a
pro-inflammatory molecule under some circumstances,15,16
although such effects were not observed in the eyes of uninfected or
recently infected mice treated with the cytokine in the present study.
Using a corneal micropocket assay, the eye acts as a convenient site to measure the activity of molecules that induce angiogenesis.18 One of the most potent angiogenesis factors (AFs) is the several isoforms of VEGF.22,23,29 In our studies, HSV-1 ocular infection was shown to induce VEGF production. However, it is not clear how HSV infection results in VEGF production, or if VEGF is the only, or even the predominant AF induced after HSV infection. Most likely, in fact, multiple AFs are involved, many of which could derive from inflammatory cells such as neutrophils that promptly invade the stroma of HSV-infected eyes.7,8 Because the corneal tissue thickens during HSK, this may cause hypoxia, also a known stimulus for angiogenesis.30,31 Identification of additional candidate AFs involved in HSV-induced angiogenesis, as well as their cellular source, requires further investigation. Some other herpesviruses can be involved in aberrant angiogenesis but this may result from the virus encoding molecular mimics of known angiogenesis factors.32-34 The absence of any reported AF molecular homologues with HSV-1 virion proteins makes this mechanism seem unlikely to account for HSV angiogenesis. Moreover HSV infection of cells results in the rapid shutdown of most host-cell protein synthesis.35,36 Accordingly, HSV-infected cells themselves represent unlikely primary sources of AF production. Indeed, preliminary data were providing evidence that VEGF production in tissues after HSV infection is predominantly a paracrine event (Zheng and Rouse, submitted).
Although multiple AFs could mediate HSV-induced angiogenesis, interestingly exogenous treatment with EMAP II seemed to limit the activity of all factors involved. Furthermore, EMAP II minimized angiogenesis induced by the potent molecule VEGF as well as another well-characterized AF, basic fibroblast growth factor.23 Accordingly, the mechanism by which EMAP II achieves anti-angiogenesis might be global. In fact, the present results, as well as some previous reports,10,11 indicate that EMAP II causes apoptosis in stimulated vascular endothelial cells. In addition, as shown in this report, EMAP II also caused apoptosis of corneal neovessels in HSV-infected eyes. Such an effect likely served to minimize the extent of new vessel growth. Nevertheless, as observed both in the VEGF-induced micropocket assay and after HSV infection, EMAP II treatment failed to abrogate angiogenesis. Moreover, once vessels were fully established we failed to observe any effect of EMAP II treatment. Conceivable, concomitant treatment with additional anti-angiogenesis molecules, especially those with a different mechanism of action, could further limit the extent of HSV angiogenesis and maybe even prevent HSK lesion expression. Experiments of this design are currently underway in our laboratory. Finally, targeting angiogenesis with appropriate inhibitors merits investigation as a management approach for human HSK.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants EY05093 (to B. T. R.) and HL60061.
Accepted for publication June 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Nature 1998, 394:485-490[Medline]
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