(Received for publication, May 19, 1995; and in revised form, July 9, 1995)
From the
Nitric oxide (NO) is an important mediator of macrophage
activities. We studied the regulation of macrophage NO synthesis by
-macroglobulin (
M), a proteinase
inhibitor and carrier of certain growth factors, including transforming
growth factor-
(TGF-
). Native
M and the
M receptor-recognized derivative,
M-methylamine (
M-MA), increased
nitrite generation by the RAW 264.7 murine macrophage cell line. The
level of nitrite accumulation, which is an index of NO synthesis, was
comparable to that observed with interferon-
. Native
M and
M-MA also increased inducible
nitric oxide synthase (iNOS) mRNA levels and substantially reduced the
number of viable cells, as determined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium/succinyl
dehydrogenase assay or trypan blue exclusion. At slightly higher
M concentrations, [
H]thymidine
incorporation was inhibited. All of these activities were counteracted
nearly completely when the iNOS competitive inhibitor N
-monomethyl-L-arginine was included. By in situ nick translation, native
M and
M-MA increased the percentage of cells with detectable
single strand chromatin nicks from 4 to 12 and 17%, respectively. This
change suggested apoptosis; however, electron microscopy studies
demonstrated variability in the morphology of injured cells. To
determine the mechanism by which
M increases
macrophage NO synthesis, we studied proteolytic
M
derivatives that retain partial activity. A 600-kDa derivative that
retains growth factor binding activity increased RAW 264.7 cell NO
synthesis and iNOS mRNA levels comparable to native
M
and
M-MA. The purified 18-kDa
M
receptor-binding fragment had no effect on NO synthesis or iNOS
expression. Thus, the growth factor-carrier activity of
M and not its receptor-binding activity is essential
for NO synthesis regulation. A TGF-
-neutralizing antibody mimicked
the activity of
M, increasing RAW 264.7 cell NO
synthesis and decreasing cellular viability. These studies demonstrate
that
M can regulate macrophage NO synthesis and
profoundly affect cellular function without gaining entry into the cell
and without binding specific plasma membrane receptors.
Enzymes of the nitric oxide synthase (NOS) ()family
catalyze the formation of nitric oxide (NO) and citrulline from L-arginine, O
, and NADPH(1, 2) .
The constitutive NOS isoforms (NOS-1 and NOS-3) produce low levels of
NO for several minutes in response to agonists that elevate
intracellular [Ca
](1) . By
contrast, the inducible isoform of NOS (NOS-2 or iNOS) generates large
amounts of NO over a prolonged period of time and does not require
Ca
-elevating agonists for activity(3) . iNOS
expression has been observed in many cells, including murine
macrophages(4, 5, 6, 7) ,
hepatocytes(8, 9) , endothelial cells(10) ,
smooth muscle cells(11) , cardiac
myocytes(12, 13) , keratinocytes(14) , and
human monocytes/macrophages that are HIV-infected (15, 16) or exposed to cancer cells(17) .
Cellular levels of iNOS are regulated by cytokines, bacterial
products, and other extracellular mediators. In murine macrophages,
interferon- (IFN-
) and bacterial lipopolysaccharide
significantly increase iNOS mRNA levels and NO
production(18, 19, 20) . By contrast,
transforming growth factor-
(TGF-
) isoforms 1, 2, and 3
decrease NO production in murine macrophages(21, 22) ,
smooth muscle cells(23, 24) , and cardiac
myocytes(13) . TGF-
1 inhibits iNOS gene transcription in
smooth muscle cells(25) . In murine macrophages, TGF-
1
acts post-transcriptionally, decreasing iNOS mRNA stability and
translation while increasing degradation of the synthesized
enzyme(22) .
NO is a potent mediator of many macrophage activities including the cytolytic response to tumor cells and microorganisms(18, 26, 27) . NO also regulates processes involved in atherogenesis(28, 29, 30, 31, 32) . When induced at high levels, iNOS can generate NO at concentrations that are cytotoxic to the cells that synthesize it and to neighboring cells(29, 33) . High levels of NO induce changes suggestive of apoptosis in mouse peritoneal macrophages and in the RAW 264.7 mouse macrophage cell line (33, 34, 35) . Thus, regulation of NO synthesis may be important under normal physiologic conditions and in various pathophysiologic states.
Human
-macroglobulin (
M) is a large (M
718,000), homotetrameric glycoprotein
found at high concentration in the plasma (2-5 µM)
and in extravascular spaces(36, 37) . Classically,
M has been described as a broad-spectrum proteinase
inhibitor; however, more recent studies suggest an important role for
M in the regulation of cellular growth and
physiology(37, 39, 40) .
M
exists in at least two well-described conformations. The native form
expresses proteinase-inhibitory activity but is not recognized by
cellular receptors(36) . ``Activated''
M is generated by reaction with proteinases or small
primary amines that modify the
M thiol ester bonds.
The structure of
M after reaction with proteinase or
methylamine (
M-MA) is equivalent, allowing the use of
M-MA as a model of the activated
M
conformation(41, 42) .
M-MA and
M-proteinase complexes are recognized equivalently by
M-specific cellular receptors. One such receptor has
been purified and characterized; this receptor is identical to low
density lipoprotein receptor-related protein
(LRP)(43, 44, 45, 46) .
M may regulate cellular growth and physiology by at
least two mechanisms, the first of which involves cytokine carrier
activity. TGF-
1, TGF-
2, platelet-derived growth factor BB,
nerve growth factor-
, and interleukin-1
are among a growing
list of cytokines reported to bind to
M(39, 40, 47) . All of these
cytokines, with the exception of TGF-
2, demonstrate higher
affinity for the activated
M
conformation(47, 48) . Nevertheless, due to the large
excess of native
M compared with activated
M in plasma and serum-supplemented cell culture
medium, native
M is frequently responsible for the
cytokine carrier activity observed in biological
systems(39, 49, 50, 51) . Many
cytokines, including IFN-
, do not bind
M with
significant affinity(47) . Therefore,
M may
alter the balance of cytokines within the microenvironment surrounding
responsive cells. Binding of TGF-
isoforms to
M
neutralizes the activity of the TGF-
toward various cells
including endothelium(51) , keratinocytes(52) , and
mink lung epithelium(49) . The extent of TGF-
neutralization correlates with the affinity of the
M/growth factor
interaction(39, 47, 51) . The growth factor
binding activities of tetrameric
-macroglobulins from different
species, including human, mouse, and rat, are comparable(39) . (
)
The second mechanism by which M may
regulate cellular function is independent of cytokine-carrier activity.
Misra et al.(53, 54) proposed that mouse
peritoneal macrophages express a second
M
``signaling receptor.'' The receptor recognizes only
activated
M but is distinct from LRP. In response to
activated
M, the second
M receptor
initiates signal transduction responses, including rapid
phosphatidylinositol 4,5-bisphosphate
hydrolysis(53, 54) . Activated
M has
been shown to affect superoxide anion production (55) and
prostaglandin E
synthesis (56) in mouse peritoneal
macrophages. The mechanism by which activated
M causes
these changes has not been fully explored.
Macrophages and
fibroblasts synthesize M(36) . Furthermore,
with loss of vascular integrity,
M might be expected
to reach high concentrations in the interstitial spaces (37) .
Therefore, we hypothesized that
M may be a significant
regulator of macrophage activity at sites of infection or inflammation,
in the intima of an injured artery, and in a vascularized tumor. The
present study was undertaken to determine whether
M
regulates macrophage NO production. Our results demonstrate that
M increases iNOS levels in RAW 264.7 cells, causing
cellular death that is prevented by a specific competitive iNOS
inhibitor. Regulation of iNOS was entirely due to the ability of
M to bind growth factors secreted by the macrophages.
Furthermore, the activity of
M was mimicked by
TGF-
-specific neutralizing antibody. Based on these studies, we
propose that
M may be a component of an important
macrophage autocrine regulatory pathway involved in controlling
cellular NO production.
Native M was purified from
human plasma according to the method of Imber and Pizzo(57) .
Final preparations were free of conformationally modified forms as
determined by nondenaturing polyacrylamide gel electrophoresis and
SDS-polyacrylamide gel electrophoresis.
M-MA was
prepared by reacting native
M with 200 mM methylamine in 50 mM Tris-HCl, pH 8.2, for 6 h at 22
°C. Unreacted methylamine was removed by dialysis against 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS) at 4 °C.
Native
M and
M-MA preparations
contained less than 0.1 ng/ml of endotoxin as determined by the limulus
lysate assay (Associates of Cape Cod, Inc., Woods Hole, MA).
M-MA was digested with papain according to the
method of Sottrup-Jensen et al.(58) , as modified by
Hussaini et al.(59) . The resulting products,
including the 18-kDa receptor-binding fragment (RBF) from the C
terminus of each
M subunit and the truncated 600-kDa
M-MA derivative were purified by chromatography on
Ultrogel AcA-22. Purified RBF competed with
M-MA for
specific binding to RAW 264.7 cells, as expected since RBF retains the
M receptor-recognition site(58) . Specific
I-
M-MA binding was completely inhibited
by 5 µM RBF; the IC
was 250 nM. The
600-kDa derivative does not bind to
M receptors but
retains growth factor binding activity (60) . RBF completely
lacks growth factor binding activity.
RAW 264.7 DNA synthesis was
assessed by [H]thymidine incorporation. The cells
were plated at 10
/well in 24-well plates and cultured first
in serum-supplemented medium for 24 h and then in SFM for an additional
24 h. At the beginning of the third day,
M-MA was
added and the cells were cultured for 24 h.
Thymidine (1 µCi/ml) was included during
the last 6 h. Cells were then washed with Earle's balanced salt
solution, 10 mM HEPES, pH 7.4, and fixed in 10%
trichloroacetic acid. Cell-associated radioactivity was recovered by
incubation in 1.0 M NaOH for 12 h. The pH was neutralized with
1 M HCl. Cell extracts were then combined with Ready-Safe
scintillation fluid for counting in a Beckman scintillation counter.
RAW 264.7 cell viability was determined by trypan blue exclusion.
Cells were plated at a density of 5 10
/well in
6-well plates and cultured for 48 h. Native
M (280
nM),
M-MA (0.14-280 nM), the
600-kDa derivative (200 nM), and RBF (200 nM) were
then added. After an additional 24-h incubation, the cells were
released, either with trypsin/EDTA (Life Technologies, Inc.) or with a
cell scraper, incubated with trypan blue, and counted using a
hemocytometer. Since the monolayers were not washed prior to
trypsin-EDTA treatment or scraping, adherent cells, and cells that
detached during incubation with
M were detected.
Figure 1:
Nitrite
generation by RAW 264.7 cells incubated for 24 h with native
M (280 nM),
M-MA (280
nM), or IFN-
(10 ng/ml). The same concentrations were
used when IFN-
and native
M or
M-MA were added to cultures simultaneously. The bars represent the mean ± S.E. of four
experiments.
Native M and
M-MA increased iNOS
mRNA levels in RAW 264.7 cells, as determined by Northern blot analysis (Fig. 2). The increase in iNOS mRNA caused by
M-MA was either equivalent to the increase caused by
native
M or slightly greater in six separate
experiments. In the particular study shown in Fig. 2, nitrite
levels were measured in the cultures from which mRNA was harvested. The
nitrite levels, like the iNOS mRNA levels, were increased similarly by
native
M and
M-MA.
Figure 2:
Northern blot analysis of iNOS mRNA and
phosphoglyceraldehyde dehydrogenase (PGAD) mRNA from cultures
treated for 24 h with 280 nM M-MA (lane
b), 280 nM native
M (lane c) or
10 ng/ml IFN-
(lane d). The control culture (lane
a) was incubated for 24 h in SFM without
M or
IFN-
. Nitrite levels were determined in the conditioned media of
the cultures from which the RNA was isolated. These levels are shown
immediately below the blot.
The increase in
iNOS mRNA caused by native M or
M-MA
was significantly less than the increase observed with IFN-
(10
ng/ml) even though all three agents increased nitrite levels similarly
(as shown in Fig. 1and Fig. 2). Thus, while the increase
in NO synthesis caused by
M is at least partially
explained by the increase in iNOS mRNA, a second contributing mechanism
may be involved. Feasible second mechanisms include an increased rate
of iNOS mRNA translation and/or stabilization of the synthesized
enzyme.
To test this hypothesis, two
M derivatives with different activities were studied.
The 600-kDa derivative retains growth factor binding activity but is
not recognized by
M
receptors(58, 60) . RBF does not bind growth factors (60)
but interacts with LRP and the second
M signaling receptor described by Misra et
al.(53, 54) . The RBF/second
M
signaling receptor interaction apparently causes the full spectrum of
signal transduction responses observed with activated
M. Fig. 3shows that the 600-kDa derivative
increased NO synthesis in RAW 264.7 cells. The extent of the response,
in the presence and absence of IFN-
, was comparable to that
observed with
M-MA. By contrast, RBF had no effect on
NO synthesis. The 600-kDa derivative also increased the level of iNOS
mRNA while RBF had little or no effect (Fig. 4). These results
confirm that the ability of
M to increase NO synthesis
in RAW 264.7 cells results from
M-cytokine
interactions occurring in the medium and not from an
M
receptor interaction.
Figure 3:
Nitrite levels in the conditioned media of
cultures treated for 24 h with the 600-kDa derivative (200 nM)
or RBF (200 nM). Of these two M derivatives,
only RBF is recognized by
M receptors while the
600-kDa derivative retains growth factor binding activity. Each
M derivative was added to cultures alone or in
combination with IFN-
(10 ng/ml). Each bar shows the mean
± S.E. of four experiments.
Figure 4: Northern blot analysis of iNOS mRNA and phosphoglyceraldehyde dehydrogenase (PGAD) mRNA from cultures treated for 24 h with 200 nM 600-kDa derivative (lane b) or 200 nM RBF (lane c). The control culture (lane a) was incubated in SFM for 24 h.
Figure 5:
Viable
cell number, as determined by MTT assay, in cultures treated for 24 h
with M-MA (280 nM), native
M
(280 nM), the 600-kDa derivative (200 nM), or RBF
(200 nM). Results were standardized by comparison with
cultures incubated in SFM for 24 h. The bars represent the
mean ± S.E. of four experiments.
To determine if the observed decrease in RAW
264.7 cell number was due to a change in the rate of proliferation or
altered cellular viability, [H]thymidine
incorporation and trypan blue exclusion experiments were performed.
Increasing concentrations of
M-MA were added to the
cultures. By MTT assay, an
M-MA
concentration-dependent decrease in the number of viable cells was
apparent (Fig. 6). This decrease was accompanied by a
significant increase in the fraction of cells that did not exclude
trypan blue in cultures treated with 10-300 nM
M-MA. [
H]Thymidine
incorporation was also decreased by high concentrations of
M-MA; however, unchanged or slightly increased
[
H]thymidine incorporation was observed with
1.4-10 nM
M-MA. Thus,
M-MA increases the fraction of RAW 264.7 cells
undergoing cell death and, at higher concentrations, decreases the
fraction of cells progressing through S-phase of the cell cycle.
Figure 6:
Viable cell number, percent viability, and
[H]thymidine incorporation in cultures treated
for 24 h with different concentrations of
M-MA. In panel A, viable cell number was determined by MTT assay
(points represent the mean ± S.E. of three experiments). In panel B, percent viability represents the fraction of cells
that exclude trypan blue (points represent the mean ± S.E. of
three experiments, each with duplicate determinations). In panel
C, [
H]thymidine incorporation was measured
(points represent the mean ± S.E. of two experiments with three
determinations/experiment).
Cultures were treated with native M, RBF, or the
600-kDa derivative for 24 h and studied by trypan blue exclusion. The
M derivatives that decreased cell number by MTT assay
(as shown in Fig. 5) also decreased the fraction of cells that
excluded trypan blue (Table 1). By contrast, RBF did not decrease
trypan blue exclusion. Equivalent results were obtained whether the
cells were released from the wells by scraping or with trypsin/EDTA.
Figure 7:
Effects of the iNOS inhibitor NMMA on the
cytotoxic activity of M. RAW 264.7 cells were
incubated for 24 h with 280 nM
M-MA (
),
280 nM native
M (
), 280 nM
M-MA + 10 ng/ml IFN-
(
), or SFM
without
M or IFN-
(
). NMMA was included at
the concentrations shown. Nitrite level measurements demonstrated that
NMMA inhibited NO synthesis in the cultures (panel A). Panel B shows the effects of NMMA on viable cell number as
determined by MTT assay. Each graph represents duplicate experiments
with quadruplicate determinations in each.
Figure 8:
ISNT studies of RAW 264.7 cells.
Representative photomicrographs are shown for cultures incubated for 24
h in SFM (panel A), 280 nM M-MA (panel B), and 280 nM native
M (panel C). Dark staining of nuclei indicates the presence of
single strand nicks in the chromatin. The bar represents 20
µm.
DNA
fragmentation was detected in some cells of the control preparation by
ISNT. Cells in the M and
M-MA-treated
cultures were labeled as well. The percentage of adherent cells that
stained positively was increased after
M or
M-MA treatment (Table 2), even though many of
the cells that were injured or dead had already detached from the
coverslips. DNA was isolated from RAW 264.7 cells treated with native
M or
M-MA for 24 h and analyzed by
agarose gel electrophoresis. Internucleosomal DNA fragmentation was not
detected (data not shown). Detection of DNA ladders provides strong
evidence for apoptosis; however, the absence of DNA ladders does not
preclude apoptosis in part or all of a cell
population(68, 69) .
Figure 9:
Representative electron micrographs of RAW
264.7 cells from control cultures (panel A) and from cultures
incubated for 24 h with 280 nM M-MA (panel B). Most of the cells in the untreated cultures had
centrally located, round nuclei with prominent nucleoli and uniform
morphology. Many of the cells treated with
M-MA
displayed various morphologic changes suggesting injury/death. The arrow marks a cell with changes suggestive of apoptosis. The arrowhead marks a cell with predominantly cytoplasmic changes,
such as dilated organelles and discontinuity of the plasma membrane. An
enlarged cell is marked with the asterisk. The bar represents 2.45 µm.
Figure 10:
TGF--neutralizing antibody mimics
the activities of
M. RAW 264.7 cells were treated for
24 h with either
M-MA (280 nM) or
TGF-
-neutralizing antibody (50 µg/ml). After 24 h, nitrite
levels were measured in the culture media (panel A). The
number of viable cells was assessed by MTT assay (panel B).
The bars represent the mean ± S.E. of two separate
experiments with quadruplicate
determination/experiment).
Conditioned medium from RAW 264.7 cells was analyzed for
TGF- activity using the FBHE proliferation assay. The conditioned
medium inhibited [
H]thymidine incorporation by
the FBHE cells, as expected for a TGF-
-containing vehicle.
Antibody 1D11.16 neutralized the activity in the conditioned medium,
confirming that the FBHE growth inhibition was due to TGF-
. The
concentration of TGF-
in the RAW 264.7 cell conditioned medium, as
determined by comparison to purified TGF-
2, was 9 ± 4
pM (n = 4).
The growth factor-carrier activity of M was
originally identified in studies of whole plasma.
O'Connor-McCourt and Wakefield (73) demonstrated that
nearly all of the TGF-
1 in plasma is associated with
M and Huang et al.(74) showed that
TGF-
1 is inactive while bound to
M. Based on
these early studies,
M
TGF-
complex was
initially referred to as latent TGF-
, a term subsequently reserved
for the precursor form of TGF-
secreted by cells in complex with a
latent TGF-
binding protein(75) . More recent studies have
shown that TGF-
1(39, 50) , platelet-derived
growth factor-BB(39) , and nerve growth factor-
(
)bind to native
M when injected
intravascularly in mice. After an initial rapid clearance phase, the
complex with
M becomes the primary form of each growth
factor present in the plasma. The
M
growth factor
complex is relatively stable in the blood, presumably forming a pool of
slowly releasable activity since complexes of growth factors with
native
M remain primarily noncovalent and reversible.
These animal model and whole plasma experiments demonstrate that
M-growth factor interactions are physiologically
significant in vivo.
In this study, we demonstrated that
M is an important regulator of RAW 264.7 cell NO
synthesis. The underlying mechanism depends on the growth
factor-carrier activity of
M. Since the experiments
were performed in serum-free medium and an alternative source of
cytokines was not provided, the critical interaction must have involved
M and one or more cytokines secreted by the RAW 264.7
cells. For a number of reasons, we examined the possible role of the
TGF-
superfamily in the
M/NO-regulatory system.
First, TGF-
isoforms inhibit macrophage NO synthesis(21) .
Furthermore, it is known that monocytes-macrophages synthesize
TGF-
(70, 71, 72) , a result confirmed
for RAW 264.7 cells in this study. Since TGF-
decreases iNOS mRNA
translation and destabilizes the protein(22) , a neutralizing
interaction of
M with RAW 264.7 cell TGF-
is
consistent with our observation that
M elevates NO
synthesis disproportionately with iNOS mRNA. Finally, we have shown
that the TGF-
-neutralizing activity of
M in FBHE
cultures closely correlates with the binding affinity
(1/K
) of the
M/TGF-
interaction (51) . We assume that the same principle holds for
macrophage cultures. Since native
M was as active, or
nearly as active, as
M-MA in the regulation of NO
synthesis, the involvement of TGF-
2 is suggested; TGF-
2 is
the only growth factor studied to date that binds native
M and
M-MA with equal affinity.
To
test the hypothesis that TGF--binding accounts for the NO
synthesis induced by
M, we studied a
TGF-
-neutralizing antibody. The antibody completely mimicked the
activity of
M, increasing cellular NO synthesis and
decreasing cellular viability. Although the spectrum of cytokines that
interact with
M in the macrophage culture medium may
be complex, the antibody studies allow us to conclude that TGF-
binding is sufficient to account for the activities of
M observed here. Furthermore, these antibody studies
identify an important autocrine regulatory loop for the RAW 264.7
macrophage cell line. Apparently, TGF-
synthesized by the cells
themselves functions to suppress iNOS expression. By interrupting this
autocrine pathway, extracellular mediators, such as
M
or antibody, can profoundly affect cellular phenotype and function
without gaining entry into the cell and without binding to plasma
membrane receptors.
As a result of the shift in available cytokines
in the culture medium, M-treated RAW 264.7 cells
underwent cell death. One possible explanation for this result is that
M withdraws a growth factor(s) that is otherwise
available to the macrophages and necessary for continued growth.
Alternatively, cell death might be mediated directly by NO, synthesized
at cytotoxic levels in the
M-treated cultures. These
two mechanisms are similar since both involve
M
interacting with macrophage-secreted cytokines. Furthermore, the
mechanisms are not mutually exclusive. To isolate the role of NO
synthesis in cell death, among other changes in cellular function that
may result from altered cytokine availability, we performed experiments
with a specific, competitive iNOS inhibitor, NMMA. Inhibition of iNOS
substantially reversed the loss of cellular viability in
M-treated cultures, demonstrating that NO is
completely, or nearly completely, responsible for death of the
macrophage cell line under these experimental conditions.
It has
been reported that macrophages, which are induced to secrete high
levels of NO by treatment with IFN- and lipopolysaccharide,
undergo apoptosis(33, 34, 35) . Therefore, we
performed ISNT and agarose gel electrophoresis experiments to examine
the changes occurring in
M-treated RAW 264.7 cells.
The ISNT experiments revealed evidence of single-stranded DNA
fragmentation, at the single-cell level, in an increased percentage of
the cells treated with native
M or
M-MA. The DNA-agarose gel electrophoresis experiments
were negative. ISNT is more sensitive than agarose gel electrophoresis
in detecting DNA fragmentation, especially when the change is limited
to a subpopulation of the cells under examination(65) .
However, labeling of cells by ISNT may detect modes of cell death other
than apoptosis. Therefore, we further explored the morphologic changes
occurring in
M-treated RAW 264.7 cells by electron
microscopy. These studies showed a heterogeneous pattern of
ultrastructural changes in each
M-treated preparation.
While some cells showed morphologic changes suggesting apoptotic cell
death, other injured and dying cells did not. One possible explanation
for this result is that the cells are at various stages of dying;
however, we believe that dissynchrony in our cultures does not entirely
explain the difference in our results and the previously reported
studies(33, 34, 35) . A second explanation is
that the various agents used to induce NO synthesis
(
M, IFN-
, and lipopolysaccharide) have different
effects on the manner in which the macrophage responds to high levels
of NO.
Since NO has been implicated in many normal physiologic and
pathophysiologic processes, studies on the regulation of NO synthesis
have broad relevance. The experiments performed here identify
M as a regulator of macrophage NO synthesis for the
first time. Additional work will be necessary to determine whether our
model cell culture system is representative of monocytes and
macrophages in vivo. Our results implicating
M in the regulation of NO synthesis are unique
compared with many previous studies that focused on the biological
activities of
M, since, in our system, native
M was active in addition to the activated form
(
M-MA). Due to the efficiency of the LRP clearance
mechanism, concentrations of activated
M are very low
in the blood(36, 39, 40) . Higher levels of
activated
M may accumulate in interstitial spaces and
in non-vascular body fluids when elevated levels of active proteinase
are available locally(37) ; however, LRP may limit
concentrations of activated
M in these
microenvironments as well. By contrast, the native form of
M is not subject to receptor-mediated clearance and
therefore is stable in the presence of LRP-expressing cells, including
macrophages, smooth muscle cells and fibroblasts. Thus, native
M, due to its growth factor binding activity and high
concentration, may be the most significant form of the protein to
consider as a potential regulator of NO synthesis in vivo.