Sep 11, 1985 - 16. Wissler, J.H., and Renner, H. Inflammation, chemotropisms and morphogenesis: Novel leukocyte-derived mediators for directional growth.
Journal
SHORT
Biology
39:233-238
(1986)
COMMUNICATION
Induction Activated Alisa
of Leukocyte
E. Koch,
of Neovascularization Human Monocytes Peter
J. Polverini,
Departments of Internal Medicine (S.J.L.), Northwestern University
and S. Joseph
by
Leibovich
(A.E.K.), Pathology Medical and Dental
(P.J.P.) and Oral Biology Schools, Chicago
Neovascularization, the process of new blood vessel growth, is an important feature of many pathologic and physiologic processes. Monocytes were isolated from citrated blood buffy coat of healthy adult human donors on FicollHypaque gradients. Mononuclear cells from these gradients were fractionated on discontinuous Percoll gradients; monocyte-enriched fractions were isolated and assessed for angiogenic activity in rat corneas. Freshly isolated monocytes as well as monocytes cultured for 20 hr on fibronectin-coated collagen gels failed to stimulate neovascularization. In contrast, adherent monocytes activated with concanavalin A (25 g/ml) or endotoxin (5 g/ml) for 20 hr were found to be potently angiogenic. We conclude that peripheral blood monocytes must be activated to acquire the ability to induce new blood vessel growth, a process central to inflammation, wound healing, and tumor development.
Key words:
monocyte,
Mononuclear macrophages,
macrophage,
phagocytes, play
important
as
represented
by
roles
in host
defense
disease processes, both as scavenger secretory cells capable of modulating mesenchymal cells [13,17]. We and
Received
August
6, 1985;
accepted
Reprint requests: S.J. Leibovich, Ave., Chicago, IL 60611.
© 1986 Alan
R. Liss,
Inc.
angiogenesis,
September Department
neovascularization circulating mechanisms
monocytes and
and
tissue
inflammatory
cells with high phagocytic capacity, the proliferative capacity of lymphoid others have demonstrated an important
and as and other role for
11, 1985. of Oral
Biology,
Northwestern
University,
311 E. Chicago
234
Koch,
macrophages
in the
induction
Polverini,
and Leibovich
of microvascular
proliferation
that
occurs
in fibrovas-
cular proliferative processes, including wound repair, inflammation, and tumor growth [5,6,8-12, 14]. In this study, we have examined the ability of peripheral blood monocytes from normal adult humans to induce angiogenesis in an in vivo model system. We have found that monocytes isolated without adherence do not express angiogenic activity,
and
angiogenic
(con
that
adherence
activity.
A) or endotoxin Human
is not
Activation
results
mononuclear
per
se
a sufficient
of adherent
in the expression cells
were
stimulus
monocytes
with
of potent
isolated
from
for
either
angiogenic citrated
expression
of
concanavalin
A
activity. blood
of
ten
healthy
donors by centrifugation on Ficoll-Hypaque (Lymphoprep, Nyegaard, Oslo, Sweden) gradients. The mononuclear cells obtained from the Ficoll-Hypaque gradients were fractionated by centrifugation on discontinuous Percoll gradients (Pharmacia, Piscat
away, NJ). An isoosmotic solution of Percoll was prepared by adding one part of 10 x concentrated phosphate-buffered saline (PBS) to nine parts Percoll stock solution. This 100% Percoll stock was diluted further with 1 x concentrated PBS to obtain solutions of 40% , 50% , 54 % , 57 % , and 60% Percoll. Mononuclear cells (1 .5 x 108) were suspended in 3 ml of 60% Percoll solution and dispensed into a 15-ml centrifuge tube (Corning Glassworks, Corning, NY). The gradient was constructed by layering 3-mi volumes of Percoll in diminishing concentrations on top of the 60% Percoll solution. The tubes were centrifuged for 30 mm at 4#{176}C,1,800 rpm, in a Beckman centrifuge
TJ6-R
(Beckman
with a siliconized interfaces, washed counted.
Viability
trypan
blue
monocytes markers. St.
and
Instruments,
Pasteur (3 x) was was
determined consistently
was assessed The percentage
Louis,
made using Diff-Quick
MO)
Palo
pipet from the with Dulbecco’s
Alto,
CA).
40% and modified
by counting found
the
to
be
percentage >
in a 1-hr
incubation
period
was (Shandon, McGaw
antibodies anti-HLA-Dr CA) and examined using
Germany) in the to be monocytes
and
anti-LeuM3 Angiogenic method [3,10]. tration of 7.5
harvested
of cells
that
excluded
purity
of
harvested
99 % . The
determined. Swickley, Park, IL).
counted, and the percentage of cells that had ingested to be greater than 85% of the total cells. Cytopreparations also made and stained with either Duff-Quick solution
West judged
were
by morphology as well as by means of several monocyte of cells phagocytizing latex particles (Sigma Chemical Co.,
a Shandon H cytocentrifuge solution (American Scientific,
gated monoclonal Mountain View,
Monocytes
50% , and 50% and 54% Percoll Eagle’s medium (DMEM), and
epifluorescence by morphology
and a Leitz
Cytopreparations
were
PA), and stained At least 200 cells
with were
latex
was determined and found of the harvested cells were or with the fluorescein conju-
anti-LeuM3 Dialux 20
mode. Greater than and positive staining
(Becton-Dickinson, microscope (Wetzlar, 95% with
of the cells were both anti-HLA-Dr
antibodies. activity was assessed using a modification of a previously described Harvested cells were washed (1 X) with PBS, suspended at a concenx 106 cells per 100 s1 in 0.85% Sea-plaque agarose solution in PBS
(Polysciences Inc., Warrington, PA), and 10-il aliquots were injected into the corneas of 200-250 gm rats, 1-1.5 mm from the limbus. Following injection, corneas were observed daily with a stereomicroscope. At day 7 post-injection, before sacrifice, the rats were perfused intraarterially with colloidal carbon (Pelican, Inc., Hanover, FRG) as described were excised, thick sections
[3], providing a permanent fixed in 2% glutaraldehyde, of methacrylate-embedded
record of neovascular responses. flattened, and photographed. corneas were also prepared,
The corneas Two-micron stained with
and Angiogenesis
Monocytes
methylene response
blue-basic manifested
sprouts
toward
fuchsin, and as sustained
the
cell
from Percoll gradients described previously 10% fetal calf serum
implant
examined histologically. unidirectional ingrowth
forming
a brush-like
lipopolysaccharide, Type
Sigma V, Sigma
by digesting
the
Chemical
Chemical
collagen
Cooper Biomedical, gentamicin, followed
gels
and
also
performed.
with
The
NJ) (1
cells, DMEM
quantitative
adherent monocytes harvested no angiogenic response. An
Control
0.5 % (w/v) and
washed
Sea-plaque containing
clostridial (3 x)
harvested
the addition of coli 055:B5
collagenase
with
agarose alone, 10% FCS and
of these
harvested only one
DMEM
(Type plus
A II,
S pg/mi
in general
experiments
from fibronectin-coated example of a negative
failed
DMEM S g/m1
containing endotoxin
are
10% were
summarized
in
directly from Percoll gradients without out of 23 corneas tested. Nonactivated
of unstimulated adherent of the limbal vasculature
injections
Monocytes
or with 25 jzg/ml con cells were then harvested
Louis, MO) MO). The
Louis,
results
Table 1. Unstimulated monocytes adherence were angiogenic in
following injection normal appearance
neovascular loops and
X) with PBS. Viability, determined by the trypan was > 98 % . These cells were injected into rat corneas for potential as described above. Control corneal injections of
boiled, and frozen 25 g/ml con A,
FCS
network.
95 % air/S % C02, or with preparation of Escherichia
Co. , St.
Co. , St.
Freehold, by washing
blue exclusion technique, assessment of angiogenic washed,
A positive of capillary
were plated on fibronectin-coated collagen gels prepared as [12] at a concentration of 1 x 106 cells/mI in DMEM containing (FCS) and 5 jsg/ml gentamicin. Dishes were incubated at 37#{176}C
for 24 hr in a moist incubator, gassed with 5 /Lg/ml bacterial endotoxin (phenol-extracted (Sigma
235
monocytes without new
to elicit
angiogenic
collagen corneal
gels likewise neovascular
is shown capillary responses.
showed response
in Figure la. The growth is apparent. In contrast,
adher-
ent monocytes stimulated with either con A (25 /Lg/ml) or endotoxin (5 jsg/ml) potently induced neovascularization. An example of a positive neovascular response obtained using monocytes stimulated with endotoxin is shown in Figure lb. The sustained unidirectional growth of capillary loops and sprouts toward the cell implant (arrow) is clearly seen. Histologically, all vascularized corneas were free of inflammatory
exudate
TABLE
and
1. Angiogenic
showed Responses
some
stromal
of Pen pheral
thickening. Blood
Monocytes No.
Substance
Negative response
injected
Nonactivated
Percoll
monocytes
from
of Corneas
Positive response
21
1
Percent positive 4
gradients
Adherent
nonactivated
Adherent monocytes con A (25 g/ml) Adherent monocytes endotoxin (5 g/ml)
monocytes
13
0
0
activated
with
0
8
100
activated
with
1
13
93
12
0
0
4
0
0
10
0
0
Controls
of washed,
frozen Controls FCS Controls FCS
cells of DMEM + 10% con A (25 tg/ml) of DMEM + 10% endotoxin (5 g/ml)
+
+
boiled,
and
236
Koch,
Polverini,
and
Leibovich
a +
b +
Fig. 1. a) Negative neovascular response. Note the normal limbal vasculature. b) In contrast, this positive neovascular response shows a dense neovascular plexus consisting of small sprouts and dilated, tortuous loops that have penetrated the corneal stroma in several planes. Arrows indicate sites of implants.
Monocytes
and Angiogenesis
237
Macrophages have been implicated as mediators of the neovascular process in several studies, including wound repair, inflammation, and solid tumor development [5,6,8-12, 14]. Polverini et al [10] first showed that viable macrophages obtained from peritoneal cavities of mice and guinea pigs were able to stimulate neovascularization when
introduced
into
intraperitoneal
injection
latex
was
particles
guinea
pig
corneas.
of paraffin
required
for
Activation
of
oil or thioglycollate the
expression
macrophages
or in vitro
of angiogenic
in vivo
by
by phagocytosis
activity
of
by these
cells.
et al [2] and Thakral et al [ 14] observed that activated macrophages derived from wounds were potently angiogenic. Polverini and Leibovich [12] have demonstrated that macrophages from a chemically induced transplantable rat fibrosarcoma were also capable of inducing neovascularization. Recently, Koch et al [5] demonClark
strated
that
purified
cells
directly
larization
with
the
from
human
[5] . Since
In our corneas found activity
potently
Monocytes
derived
from
monocytes
angiogenic
potent
precursor
cells,
with
Percoll
gradients
in this system. It appears property of monocytes
the
blood-borne
whether
monocytes (5 jsg/ml)
in the normally
exogenous
and
of neovascu-
endotoxin
implanted
additional
isolated
inducers
to determine
activated
when
from
without
macrophages,
were
it important activity.
prepared
cultured
to be nonangiogenic is not an intrinsic
activated
are
adherent
were
monocytes
synovia,
[15], we thought this angiogenic
experiments,
of rats.
adherent
of differentiated
rheumatoid
macrophages
circulating monocytes also inherently express con A (25 ILg/ml)
characteristics
without
adherence
activation
were,
that expression but requires that
or
avascular and
however,
of angiogenic they first be
for its induction.
The
biochemical
angiogenic
nature
activity(s)
Leibovich
and
of
(MDAA)
Polverini
[7]
the
activated
has not
monocyte
yet been
demonstrated
that
for
mesenchymal
cells
[6,9],
macrophage-derived
in detail.
macrophage-derived
(MDGF), partially purified through high pressure chropack-GPC 100 gel filtration column, possessed which in our hands has a relatively high molecular mitogen
and
characterized liquid potent weight
stimulating
Recently,
growth
factor
chromatography on a synangiogenic activity. MDGF, (70,000-80,000) is a potent
fibroblast,
smooth
muscle,
and
endothelial cell growth. Wound fluids rich in macrophages a low molecular weight factor that is angiogenic in vivo, but not mitogenic activity for endothelial cells in vitro
factor [16].
produced MDGF
by porcine
adherent, unstimulated A, however, rapidly
demonstrated that several functional
the expression express expression activated
activity activity.
by monocytes However,
experiments
of monocytes MDGF
by
with
endotoxin
adherent
thus appears in this system,
MDGF is the active angiogenic properties of monocytes and
of activity our
in detectable
monocytes. Stimulation induces production of
Acquisition of angiogenic tion of MDGF producing
From
monocytes
is not produced
are also reported to contain while exhibiting chemotactic [1J. A low molecular weight activated with con A has also been reported quantities by nonadherent monocytes or by
agent, and macrophages
or con
monocytes
[4].
to parallel the acquisiwe have not as yet it should require
be noted activation
that for
[13]. we
conclude
that
a)
nonadherent
monocytes
do
not
angiogenic capacity, b) adherence is not per se a sufficient stimulus for of angiogenic activity by monocytes, and c) adherent monocytes can be in vitro to express angiogenic activity by incubation with con A or endotoxin.
Koch,
238 It is possible
that
tissue sites, such become activated progression
when
and
Polverini,
monocytes
migrate
Leibovich
from
the circulation
to extravascular
as inflammatory foci, healing wounds, or growing tumors, in vivo and acquire the angiogenic activity that is central
of inflammation,
wound
repair,
and
tumor
they to the
growth.
ACKNOWLEDGMENTS
This work was supported, a grant from the 3M Corporation. from
the Illinois
in part,
Dr. of the Arthritis
Chapter
by USPHS
grant
Koch was supported, Foundation.
No.
ROl-GM29 in part,
135 and by
by a fellowship
REFERENCES I. Banda, Mi., Knighton, D.R., Hunt, T.K., and Werb, Z. Isolationof a nonmitogenic angiogenesis factor from wound fluid. Proc. Nail. Acad. Sci. U.S.A. 79,7773, 1982. 2.
Clark,
R.A.,
Stone,
RD.,
Leung,
D.Y.K.,
Silver, D.C., Hahn, D.C., and Hunt, T.K. Role of macrophages in wound healing. Surg. Forum 27,16, 1976. 3. Gimbrone, G.A., Jr., Cotran, R.S., Leapman, SB., and Folkman, J. Tumor growth and neovascularization: An experimental model
using
the
52,413, 1974. 4. Glenn, K.C, and derived growth cells: Activation concanavalin-A. 5.
Koch,
A.E.,
rabbit
cornea.
J.N.C.I.
Ross, R. Human monocytefactor(s) for mesenchymal of secretion by endotoxin and Cell 25,603, 1981.
Leibovich,
Si.,
and
Polverini,
P.1. Human rheumatoid synovial macrophages induce angiogenesis. Arthritis Rheum. (Suppl.) 28,27, 1985. 6. Leibovich, Si. Mesenchymal cell proliferation in wound repair. The role of macrophages. in Soft and Hard Tissue Repair (Hunt, T.K., Heppleston, RB., Pines, E., and Rovee, D., Eds.) New York: Praeger Press, p. 329, 1984. 7.
Si., and Polvenni, P.1. Partial purification of macrophage-derived growth factor (MDGF) and macrophage-derived argiogenic activity (MDAA) by gel filtration high pressure liquid chromatography. Br. J. Rheum. 24(Suppl. 1), 197, 1985. Leibovich,
8. Leibovich, Si., and Ross, R. A macrophagedependent factor that stimulates the proliferation of fibroblasts in vitro. Am. 1. Pathol. 84,501, 1976. 9. Martin, B.A., Gimbrone, MA., Jr., Unanue, E.R., and Cotran, R.S. Stimulation of non-
lymphoid
mesenchymal
macrophage-derived
cell proliferation growth
factor.
by a
1. Immu-
nol. 126,1510, 1981. 10. Polverini, P.1., Cotran, R.S., Gimbrone, M.A., and Unanue, ER. Activated macrophages induce vascular proliferation. Nature 269,804, 1977. 11. Polverini, P.1., Cotran, R.S., and Sholley, MM. Endothelial proliferationin the delayed hypersensitivity reaction: An autoradiographic study. 1. Immunol. 118,529, 1977. Polverini, P.1., and Leibovich, S.J. Induction of neovascularization in vivo and endothelial cell proliferation in vitro induced by tumor associated macrophages. Lab. Invest. 51,635, 1984. 13. Takemura, R., and Werb, Z. Secretory products of macrophages and their physiological functions. Am. J. Physiol. 246,C 1, 1984. 14. Thakral, K.K., Goodson, W.H., III, and Hunt, T.K. Stimulation of wound blood vessel growth by wound macrophages. 1. Surg Res. 26,130, 1979. 15. Van Furth, R. Identification of mononuclear phagocytes: Overview and definitions. In Methods for Studying Mononuclear Phagocytes. (Adams, DO., Edelson, P.1., and Koren H., Eds.) New York: Academic Press, p. 12.
243,
1981.
16. Wissler, J.H., and Renner, H. Inflammation, chemotropisms and morphogenesis: Novel leukocyte-derived mediators for directional growth of blood vessels and regulation of tissue neovascularization. Hoppe Seylers Z. Physiol. Chem. 362,242, 1981. 17. Ziats, N.P., and Robertson, A.L., Jr. Effects of peripheral blood monocytes on human vascular cell proliferation. Atherosclerosis 38,401, 1981.
