Bovine Retinal Angiogenesis Factor Is a Small
Molecule (Molecular Mass < 600)
Simon F. Elsrow,* Ana M. 5chor,f and Jacqueline D. Weiss*
Two methods were used to extract angiogenic activity from bovine retina. Both methods initially gave
rise to nondialyzable (> 10,000 Mr) fractions with angiogenic activity. However, after anion exchange
chromatography, 20% of the extracts from one of the two methods (method 2) contained a small
molecule with angiogenic activity (Mr 300-600). Alcohol treatment of high molecular mass fractions
from both methods also released a low molecular mass angiogenic factor (Mr 300-600). No angiogenic
activity was left in the nondialyzable residue. Thi high molecular mass angiogenic fraction obtained
by method 1 after DEAE-cellulose chromatography contained a protein immunologically and electrophoretically identical to bovine serum albumin. The low molecular mass retinal angiogenic factor was
able to stimulate microvessel endothelial cell proliferation as well as being positive in the chick
chorioallantoic membrane test. The presence of a protein carrier system for a small angiogenesis
factor is proposed. This would explain discrepancies in the apparent molecular mass of retinal
angiogenic factors described previously. Invest Ophthalmol Vis Sci 26:74-79, 1985
There have been several recent reports describing
angiogenic activity present in retinal extracts from
different species. These reports show a discrepancy in
the size of the factor isolated by different workers. A
bovine retinal angiogenic fraction was shown, by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
to contain two components with molecular mass
50,000 and 70,000 after purification.12 More recently,
the same workers have reported the purification of
an angiogenic factor from bovine retinas with a
molecular mass of 23,500.3
In contrast, a low molecular mass, freely dialyzable
factor has been reported in feline retinas.4 The molecular mass of this latter factor was determined by
gel filtration and shown to be in the range of 300600—much smaller than other retinal factors. This
factor was purified by affinity chromatography using
an antibody raised against crude rat Walker 256
tumor angiogenesis factor (TAF).5 The same workers,
using the same antibody affinity technique, had previously isolated a similar, possibly identical, low
molecular mass angiogenic factor from rat Walker
256 tumor extracts.6
In the present report, we describe the extraction of
a low molecular mass angiogenic factor from bovine
retinas, which we have obtained without recourse to
an antibody preparation technique. During the processing of this factor, we formed the impression that
it was at some point bound to a carrier protein of
molecular mass === 70,000, which cross-reacted with
an antibody to bovine serum albumin (BSA).
Materials and Methods
Adult bovine eyes were obtained from the local
abattoir and transported to the laboratory not longer
than 1-2 hr after animal death. The retinas were
removed immediately at 4°C and extracted.
Method 1: Isotonic Extraction
This method was a modification of a previously
published method.1 Retinas were suspended in phosphate buffered saline (Dulbecco A; Oxoid Ltd, UK),
and stored at room temperature for 3 hr. The suspension was centrifuged at 600 g to remove cells and
then recentrifuged at 100,000 g for 30 min. The pellet
was discarded, and the supernatant acidified at 4°C
to pH 4.5 with 0.1 M acetic acid. The resulting
precipitate was centrifuged at 100,000 g for 30 min,
and the clear supernatant neutralized to pH 7.2 with
0.1 M NaOH, before being dialyzed against distilled
water (four changes 1:10 v/v) and freeze dried. The
precipitate was redissolved in 0.5 M NaOH dialyzed
against distilled water (four changes 1:10 v/v) and
freeze dried. Protein concentration was determined
From the Department of Rheumatology, Medical School, University of Manchester,* and the CRC Department of Medical
Oncology, Christie Hospital and Holt Radium Instituted Manchester, United Kingdom.
Submitted for publication: April 18, 1984.
Reprint requests: Jacqueline B. Weiss, DSc, Department of
Rheumatology, Medical School, University of Manchester, Oxford
Road, Manchester, M13 9PT, England.
74
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No. 1
BOVINE RETINAL ANGIOGENESIS FACTOR / Elsrow er ol.
on the cell-free supernatant and on the supernatant
after acid precipitation.7 At each stage samples were
tested on the chick chorioallantoic membrane (CAM)
for angiogenic activity.6 The test material was suspended in 10% v/v elvax solution (Ethylene/Vinyl
acetate—40% Vinyl acetate; Aldrich Chemical Co,
UK). The elvax pellets were washed in 100% ethanol
and dissolved in dichloromethane (BDH, UK, Analar
reagent). Pellets (1 mm 2 ) were cut and applied to the
CAM. Each sample was tested on three eggs simultaneously and membranes with visible signs of inflammation such as cell accumulation or oedema
were discarded and the sample retested. Angiogenic
activity was graded as negative, weakly positive, and
positive.
75
Sodium Dodecyl Sulphate Polyacrylamide
Gel Electrophoresis (SDS-PAGE)
SDS-PAGE was carried out by standard methodology.8 Gels were stained with 0.25% Coomassie
Brilliant Blue R250 in 25% propan-2-ol/10% acetic
acid/water (v/v/v) and scanned at 580 nm using a
Pye Unicam SP8100 spectophotometer with densitometer attached.
Immunodiffusion Assay
Immunodiffusion was carried out by the Ouchterlony technique. Antibody was placed in the center
well of the plate, and test samples and known controls
in the surrounding wells.
Method 2: Homogenization
Further Treatment of High Molecular Weight
CAM +ve Material
Retinas were homogenized in batches of 10-20
using the method employed to homogenize feline
retinas.4 Protein concentration was determined on
the crude extract as before, and all fractions were
tested on the CAM.
The freeze dried supernatants from method 2 were
suspended in 100% ethanol (10 mg/ml) and stirred at
4°C for 2 hr before centrifugation at 10,000 g for 15
min. The supernatant was decanted and the pellet
reextracted with 100% ethanol. The two ethanol
supernatants were pooled, rotary evaporated under
reduced pressure to a small volume, water was added
and reevaporated to ensure removal of all the ethanol.
The wet residue was dissolved in 10% (v/v) Propan2-ol/water. Alternatively, the freeze-dried supernatants
from method 1 were suspended in 10% Propan-2-ol/
water (v/v) (5 mg/ml) and heated to 56°C for 30 min
prior to centrifugation at 50,000 g for 20 min. Pellets
from both extraction methods were redissolved in a
minimum volume of distilled water, sterile filtered,
and freeze dried. The supernatants from both extraction methods were applied to Biogel P2 columns as
described previously and the effluent was tested on
the CAM.
DEAE-Cellulose Chromatography
Twenty-five-milligram samples of freeze dried extracts were dissolved in 2 ml of 0.05 mM NH 4 HCO 3
pH 7.9 and applied to a column ( 1 X 8 cm) of
DEAE-cellulose (DE 52, Whatman) previously equilibrated with the same buffer. The column was eluted
at 60 ml/hr and monitored at 206 nm or 280 nm
(Uvicord S; LKB, Sweden). Twelve milliliter fractions
were collected.
Bound material was eluted with a NaCl gradient
in 0.05 mM NH 4 HCO 3 pH 7.9 either stepwise (0.1
M, 0.3 M, 0.5 M, and 1.0 M NaCl) or with a convex
gradient (0-0.3 M NaCl) followed by two stepwise
elutions at 0.5 M and 1.0 M NaCl. All fractions were
sterile filtered, freeze dried, and tested on the CAM.
Biogel P2 Chromatography
Fractions obtained from DEAE-cellulose chromatography, which had angiogenic activity on the CAM,
were desalted on a column (4.4 cm X 44 cm) of
biogel P2 (Biorad, UK) in 10% Propan-2-ol/water
(v/v), which was eluted with the same solution.6
Eluant was monitored at 206 nm. Fractions obtained
were rotary evaporated under reduced pressure until
the volume was approximately 10 ml. As an inert
bulking agent, 200 n\ of a 50 mg/ml lactose solution
(Sigma) were added to each sample. Fractions were
sterile filtered, freeze dried, and retested on the CAM.
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Endothelial Cell Proliferation In Vitro
Retinas from two to four freshly dissected bovine
eyes were chopped freely with scissors and incubated
first with bacterial collagenase EC 3.4.24.3, (0.5 mg/ml;
Millipore Ltd, UK) for 16-20 hr and secondly with
trypsin EGTA for 15 min (0.05% Trypsin EC 3.4.21.4
(Sigma Chemical Co; UK) with 2 mM EGTA). The
resulting homogenate was centrifuged at 120 g for 5
min, the residue resuspended in tissue culture medium
and plated onto gelatin-coated tissue culture dishes. The
medium used was Eagles' Minimum Essential Medium
(MEM) with 15% donor calf serum (DCS), 2 mM
Glutamine, 1 mM Sodium Pyruvate, nonessential amino
acids (Gibco Bio-Cult; Europe Ltd.) and 50 mg/ml
ascorbic acid (15% DCS-MEM).
76
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1985
Vol. 26
Table 1. Protein concentrations and CAM results
for crude extracts of bovine retinas
Number
of
extracts
Extract
Crude isotonic
extract (method 1)
Acid soluble fraction
from isotonic
extract
Crude homogenised
extract (method 2)
Ion exchange
chromatography
of isotonic extract
(method 1)
Ion exchange
chromatography
of homogenized
extract (method 2)
14
Protein
concentration
mg/retina
7.8 ±
CAM result
+ve
-ve
3
11
3.4
I
03
12
10
2.2 ±
1.5
18.6 ± 11
6
6
0
10
11
Nd
8
3
17
Nd
16
1
Two to 3 days later, the medium was discarded,
the cultures washed five times with fresh medium,
and left incubating with fresh medium. Various types
of isolated cells and colonies were present in the
primary cultures. Endothelial cell colonies were identified by their typical cobblestone morphology. Those
colonies were ring cloned and cultured as described
earlier.9 Endothelial origin was confirmed by immunolocalization of factor VIII antigen,10 as well as by
their typical morphology and growth characteristics.12
Two-milliliter aliquots of a cell suspension containing 0.6-1 X 105 cells were plated onto collagen or
gelatin coated dishes and incubated at 37°C in a
humidified atmosphere of 5% CO 2 95% air. The next
day the number of adherent cells was determined,
and the medium were changed. The test sample was
dissolved in 50-100 ix\ of of serum-free MEM and
added to the cultures, while an equal volume of
MEM was added to the controls. Medium was
changed every 2 days where experiments lasted longer
r
Method i
1
06 I
\
1
i
\
\
__]rv
Fig. 1. B, DEAE-cellulose chromatography profile of retinal
homogenate. Angiogenic activity eluted in fraction IV or fraction
V. On two occasions, weak angiogenic activity eluted in fraction
II. G: Start of gradient. Bars represent fractions collected. Broken
line represents the NaCl gradient.
than 3 days. Cell harvesting and determination of
cell numbers was the same as described before.9 The
significance of differences between means was estimated by the Student's t-test.
Adult bovine aorta endothelial cells and microvessel
endothelial cells from cow brain white matter were
isolated as described previously." 12
Results
Crude Extracts
The protein content and angiogenic activity (measured by CAM test) of the crude extracts prepared by
either method 1 or 2 are shown in Table 1. Method
2 extracted a higher average concentration of protein
per retina than method 1. However, no obvious
increase in angiogenic activity was observed although
the CAM test cannot really be considered as a quantitative assay.
Acid precipitation of the isotonic extract removed
over 75% of the protein originally extracted and also
increased the number of samples that showed angiogenic activity, as reported previously.2
The diffusates of the isotonic extracts (method 1)
and the diafiltrates of the homogenized extracts
(method 2) did not exhibit angiogenic activity in the
CAM. The acid-insoluble fractions from method 1
were also nonangiogenic on the CAM.
/\
_JXJ^
J
Fig. 1. A, DEAE-cellulose chromatography profile of retinal
isotonic extract after acid precipitation. Angiogenesis activity eluted
in fraction II. Occasionally weak angiogenic activity eluted in
fraction V.
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1
DEAE-Cellulose Chromatography
Figure 1 shows typical DEAE-cellulose chromatography elution profiles for both isotonic (method 1)
and homogenized (method 2) samples. For both
profiles, fraction 1 contained the unbound material,
while all other fractions were bound. Angiogenic
activity was measured on the CAM.
No. 1
77
DOVINE RETINAL ANGIOGENESIS FACTOR / Elsrow er ol.
Acid-soluble material from method 1 showed an
angiogenic response in fraction II (0,1 M NaCl wash)
and occasionally a weak positive response in fraction
V (1.0 M NaCl wash).
Material from method 2 showed angiogenic activity
in either fraction IV (0.5 M NaCl wash) or fraction
V (1.0 M NaCl wash). On two occasions, weak positive
activity was observed at the beginning of the convex
gradient in fraction II (approximately 0.1 M NaCl).
After ion exchange chromatography, the number of
samples that were positive on CAM test increased
(Table 1).
Angiogenic fractions were purified further by extensive dialysis against distilled water or by gel filtration using P2 Biogel. Figure 2 shows the typical
elution profile from an angiogenic sample applied to
P2 Biogel.
After dialysis, the angiogenic fractions from DEAE
cellulose chromatography of isotonic extracts were
found to be in the retentate and therefore had a
molecular weight greater than 10,000.
Angiogenic fractions from homogenized extracts
after DEAE-cellulose chromatography eluted either
in the void volume (80% of the fractions tested) or
in the included region molecular weight 300-600
(20% of the fractions tested).
Chick Chorioallantoic Membrane Response
Figure 3 shows a positive angiogenic response on
the chick chorioallantoic membrane test. The elvax
pellet contains a fraction from DEAE cellulose chromatography. Also shown is a negative control in
which the elvax pellet contains only NaCl. A positive
response is characterized by the bending of medium
sized and small blood vessels towards the site of the
angiogenic sample, forming a characteristic "spoke
wheel" pattern.
Fig. 2. Typical P2 Biogel elution profile for the retinal extracts
after DEAE-cellulose chromatography. Bar indicates the point of
elution of the low molecular weight angiogenic fraction.
angiogenic fraction was in the 0.5 M NaCl (track 6).
A standard sample of BSA is electrophoresed in track
8 (unreduced) and track 9 (reduced). No components
were observed in the 0.5 M NaCl wash. This was
confirmed by scanning the gel at 580 nm (data not
shown). The angiogenic fraction shown in Figure 4B
eluted in the void region of the P2 column and
therefore was not in the low molecular weight form.
The presence of bovine serum albumin in the
active high molecular mass material obtained by
method 1 was confirmed by immunodiffusion (Figure
5). A precipitin line between antiserum to BSA and
the test sample can be seen clearly.
Further Treatment of High Molecular Weight
Angiogenic Fractions
Crude extracts previously shown to contain angiogenic fractions of molecular mass greater than 1,000
yielded low molecular mass (300-600) material after
treatment with either 10% propan-2-ol/-water (v/v)
at 56°C or 100% ethanol at 4°C. However, after gel
SDS-PAGE
Fractions from both methods 1 and 2 after DEAEcellulose chromatography were electrophoresed in
10% slab gels. Figure 4 shows the electrophoretograms
obtained. Fractions from an isotonic extract are shown
in Figure 4A. The angiogenic activity was in the 0.1
M NaCl wash, which was electrophoresed in the
unreduced state in track 3 and reduced (with dithiothreitol) in track 4. Also shown in tracks 8 and 9 is
a standard sample of bovine serum albumin (BSA;
Sigma) both in the unreduced and reduced form. The
component present in tracks 3 and 4 appeared to
electrophorese identically to the sample of BSA in
tracks 8 and 9 and this was confirmed by scanning
the gels at 580 nm (data not shown). Fractions from
an homogenized extract are shown in Figure 4B. The
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Chick Chorioallantoic Membrane Assay
p u b i i i v t;
control
Fig. 3. Chick chorioallantoic membrane showing, left, positive
test; right, negative control. Arrows indicate point of application of
test and control samples. Samples were marked either positive or
negative. A grading system was not employed.
78
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1985
Vol. 26
dye
front
T
1*-*
Fig. 4. Ten percent SDS-PAGE showing the fractions obtained from: A, left, isotonic extract (method 1) (I: unbound; II: 0.1 M NaCl
wash; III: 0.3 M NaCl wash; IV: 0.5 M NaCl wash; and V: 1.0 M NaCl wash) and B, homogenized extract (method 2) after DEAE-cellulose
chromatography. Roman numerals 1-V refer to the fractions obtained from the DEAE cellulose column. Also shown are standard samples
of bovine serum albumin (U: unreduced; R: reduced; O: origin of sample; BSA: bovine serum albumin; and I: unbound; II: 0-0.2 M NaCl
fraction of convex gradient; III: 0.2-0.3 M NaCl fraction of convex gradient; IV: 0.5 M NaCl wash; and V: 1.0 M NaCl wash).
> •
filtration no traces of angiogenic activity remained in
the excluded region of the column.
Microvessel Endothelial Cell Stimulation
Figure 6 shows the growth stimulation of bovine
retinal endothelial cells (CREC) and bovine brain
r
Fig. 5. Immunodiffusion plate showing cross-reaction between
the angiogenic fraction from method I and anti-bovine serum
albumin (center well) (1 = Anti BSA; 2 = Angiogenic Fraction
[method 1]; and 3 = BSA 0.1 mg/ml).
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Fig. 6. Effect of low molecular weight bovine angiogenic factor
on (1) bovine retinal endothelial cells (CREC) and (2) bovine brain
white matter endothelial cells (CBEC) grown on type I collagen.
Results are expressed as a percentage increase in cell number
compared with control cultures grown in the absence of angiogenic
factor. Retinal endothelial cells were cultured for 3 days, and white
matter endothelial cells were cultured for 4 days. Test samples were
bulked in NaCl.
No. 1
DOVINE RETINAL ANGIOGENESIS FACTOR / Elsrow er QI.
white matter endothelial cells (CBEC) grown on type
I collagen. Approximately 200-300 ng of the low
molecular weight angiogenic fraction from a single
retina, bulked in NaCl after DEAE-cellulose chromatography, achieved maximum stimulation per culture for both cell types. (Each culture had a total
volume of 2 ml). Stimulation produced a bell-shaped
response. No growth stimulation was observed when
the cells were grown on gelatin of type I collagen.
Adult bovine aorta endothelial cells did not respond
to the angiogenic fraction under the same conditions.
This is in agreement with a previous report.''
Discussion
Bovine retinas contain both high molecular weight
(>1000) and low molecular weight (300-600) angiogenic activity. Under dissociating conditions, the
low molecular weight form can be obtained from
high molecular weight material, which suggests that
the angiogenic factor is a small molecule attached to
a larger carrier molecule probably a protein. Further
evidence for the low molecular weight nature of the
angiogenic fraction came from DEAE-cellulose purification and gel nitration of material extracted by
homogenization. Furthermore, this low molecular
weight angiogenic material stimulated microvessel
endothelial cell proliferation on collagen gels in vitro
in a manner characteristic of low molecular weight
angiogenic factors from tumor sources.9
Purification of the high molecular weight angiogenic
factors obtained by the isotonic extraction method
(method 1) yielded a single component running as a
faint band on SDS polyacrylamide gel electrophoresis,
with a molecular weight 70,000. This component
behaved identically to bovine serum albumin either
in reduced or nonreduced form and was immunologically identical to bovine serum albumin. No bovine
serum albumin was found associated with angiogenic
fractions from homogenized tissue (method 2). The
angiogenic fractions isolated by method 2 only showed
angiogenic activity after anion-exchange chromatography. However, the presence of weak angiogenic
activity could be detected without anion-exchange
chromatography in six extracts isolated by method 1
after they had been acid precipitated. This would
suggest that crude retinal extracts contain components
capable of inhibiting angiogenic factors that are subsequently removed during purification. The absence
of angiogenic activity in the void volume of biogel
P2 volumns in previously active retinal fractions
extracted with either ethanol or propan-2-ol is further
evidence for a carrier system for a low molecular
weight angiogenic factor. The discrepancy in molecular weights for angiogenic factors isolated by different
workers from retinas and from other tissues is more
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79
likely due to differences in molecular weights of the
carrier molecule(s) than of the angiogenic factor(s)
themselves. A low molecular weight angiogenesis
factor has now been isolated from solid tumor, 6 1 3 1 4
tumor cell conditioned medium (in press) synovial
fluid,1516 retinas4 vitreous humor, and lymphocyte
conditioned medium (in preparation). In all these
cases as with bovine retina, the residual proteinaceous
material from which the low molecular weight angiogenic fraction was obtained had no further angiogenic
activity.
Key words: bovine, retina, angiogenesis, endothelial cell
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