Download The role of hyaluronic acid in protecting surface

Rheumatology 2001;40:336±340
The role of hyaluronic acid in protecting
surface-active phospholipids from lysis by
exogenous phospholipase A2
D. W. Nitzan, U. Nitzan1, P. Dan1 and S. Yedgar1
Department of Oral and Maxillofacial Surgery, The Hebrew
University±Hadassah School of Dental Medicine, Jerusalem and
1
Department of Biochemistry, Hadassah University Hospital,
The Hebrew University±Hadassah Medical School, Jerusalem, Israel
Abstract
Background. This in vitro study aimed to elucidate the extent and kind of involvement
of hyaluronic acid (HA) in the currently accepted view of synovial joint lubrication, in which
surface-active phospholipids (SAPL) constitute the main boundary lubricant. The integrity
of SAPL is apparently threatened by the lysing activity of phospholipase A2 (PLA2).
Methods. The effects of increasing concentrations of HA degraded by free radicals and
non-degraded HA on the lysing activity of PLA2 were examined in vitro. Liposomes (lipid model
membrane) containing phosphatidylcholine (PC) were used as the substrate, on the assumption
that they are appropriate representatives of SAPL.
Results. HA adhered to the phospholipid membrane (liposomes), inhibiting their lysis by
PLA2. However, in its degraded form, HA not only failed to inhibit PLA2-lysing activity, but
accelerated it.
Conclusions. It is reasonable to assume that HA plays an important indirect role in the steady
state of the boundary lubrication process of joints by protecting SAPL from being lysed by
PLA2. However, as excessive loading generates free radicals within the joint (among other
effects), the HA that is degraded in this way is incapable of protecting SAPL from lysis by PLA2.
When the rate of degradation exceeds that of synthesis, there will be insuf®cient replacement
of HA anduor SAPL, resulting in denudation of the articular surfaces. These are then exposed
to increasing friction, and hence increased danger of degenerative joint changes.
KEY WORDS: Hyaluronic acid, Lubrication, Phospholipase A2, Surface-active phospholipids,
Synovial joint, Degenerative joint changes.
Hyaluronic acid (HA) lends the normal human synovial
¯uid (SF) its remarkable rheological properties. Consequently, for many years HA was credited with the key
role in the boundary lubrication system of joints, and
as such was believed to occupy a primary place in the
pathophysiology and therapy of joint disorders w1±6x.
However, the role of HA within the framework of
the joint's boundary lubrication system has been questioned. HA possesses a negligible load-bearing capacity
and, moreover, degradation of the viscous HA by the
use of hyaluronidase does not have a detrimental effect
on the lubricating ability of the SF w7±9x. As a result,
an array of other possible functions of HA in joint
movement has been suggested, among which are those
of space ®ller, wetting agent, ¯ow barrier within the
synovium, and protector of the cartilage surfaces w1±6x.
Beside its mechanical role in joint function, HA has been
found, inter alia, to support joint integrity by acting as a
scavenger, inhibiting phagocytosis and chemotaxis, and
by preventing scar tissue formation and angiogenesis
w1±6, 10±15x.
It seems, however, that the multifunctionality of HA
is not the whole picture, as some aspects of the role
of HA in synovial joint boundary lubrication are still
obscure. A comprehensive understanding of its role
requires clari®cation of the currently accepted view of
the lubrication of synovial joints.
In the last decade it has been proposed that surfaceactive phospholipids (SAPL) serve as the major boundary lubricant, reducing the coef®cient of kinetic friction
to a very low value (0.001±0.006) and thus lessening wear
of the articular surfaces, even under high loads w16±20x.
The role of SAPL as a lubricant has been examined
by removing them from joints by the use of lipid
Submitted 14 January 2000; revised version accepted 9 October
2000.
Correspondence to: D. W. Nitzan, Department of Oral and
Maxillofacial Surgery, Faculty of Dental Medicine, P.O.B. 12272,
Jerusalem 91120, Israel.
336
ß 2001 British Society for Rheumatology
Hyaluronic acid protects against PLA2 activity
solvents w21x or, more speci®cally, of phospholipase
A2 (PLA2) w22x. Their removal induces a signi®cant
increase in friction.
Lubricin, a glycoprotein that has been isolated
from the load-bearing fraction of the synovial ¯uid
and has been identi®ed as a major boundary lubricant
w23, 24x, has been shown to be a water soluble-carrier
of SAPL w22x. Proteolipid, another component of the
SF, has been shown to facilitate the deposition of
the oligolamellar (graphite-like) layer of SAPL, and so
aids in boosting boundary lubrication of the articular
surfaces w20x.
When functioning under load, the boundary lubrication system adapts itself constantly by a process of
remodelling. In this process, PLA2, which is secreted by
synoviocytes, chondrocytes and osteoblasts into the synovial ¯uid w25x, is probably responsible for the lysis of
SAPL. It is therefore assumed that, when uncontrolled,
its presence in the SF constitutes a threat to the continuity of the SAPL lining. Hence, it is of interest to
establish the factor that governs the lysing activity of
PLA2 and to identify the element that protects the continuity of SAPL in the steady-state remodelling process
in such a way that the rate of lysis does not exceed the
rate of synthesis.
This study, which was undertaken in order to elucidate
the role of HA in preserving the integrity of the boundary lubricating system of synovial joints, explored two
hypotheses. The ®rst hypothesis was that high molecular weight HA inhibits PLA2 activity and thus protects the integrity of SAPL. In the event of excessive joint
loading, or during an in¯ammatory process, reactive oxidative species are generated, which are known for their
high potency in degrading HA and impeding its synthesis w14, 26x. The second hypothesis was that HA in its
degraded form (dHA) loses its ability to inhibit PLA2
activity, leaving the SAPL vulnerable to lysis by PLA2.
To examine these hypotheses, we used liposomes
(a lipid model membrane), which contain phosphatidylcholine (PC), as a substrate. We assumed that liposomes
are optimal simulators of SAPL.
Materials and methods
Liposomes made from dioleoylphosphatidylcholine
(DOPC) and 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole) aminocaproylphosphatidylcholine (C6-NBD-PC)
were purchased from Avanti Biochemicals (Alabaster,
AL, USA). Thin-layer chromatography plates were purchased from Whatman (LK6; Whatman, Maidstone,
UK). Naja mocambique phospholipase A2, hyaluronic
acid and all other laboratory chemicals and buffers
were obtained from Sigma (St Louis, MO, USA).
Degradation of HA
HA (10 mguml of 100 mM Ca2+-free Tris buffer, pH 8.0)
was exposed to free radicals by exposure to H2O2 (4 mM)
in the presence of traces of Cu2+ and Fe2+ (10 mM) for
30 min. The reaction was terminated by the addition of
catalase (200 mg) for 10 min followed by dialysis against
337
Ca2 + -free Tris buffer (pH 8.0). Degradation of HA
was monitored by the reduction in viscosity of its solution, using an Oswald capillary viscometer (Cannon
Instruments Co., State College, PA, USA).
Effect of HA and of dHA on lysis of phospholipids by
PLA2
Liposomes were used as a substrate to study their lysis
by PLA2 in the presence of either HA or dHA. A mixture
of the liposomes DOPC and the ¯uorescent C6-NBDPC (3 : 2 molar ratio) was evaporated under a stream of
nitrogen. The lipid extract was then resuspended in
Ca2 + -free Tris buffer (100 mM, pH 8.0) and ®ltered
successively through polycarbonate Millipore ®lters (0.2
and 0.4 mM). The puri®ed liposome substrates were preincubated with HA (®nal concentration 0.0±6.88 mguml)
and with dHA (®nal concentration 0.0±2.5 mguml) for
30 min in a water bath at 378C. Naja mocambique PLA2
(30 ml; 0.014 Uuml) in Tris buffer (100 mm, pH 8.0)
supplemented with Ca2 + (10 mM) was then added to
the mixture to a ®nal volume of 800 ml. Alternatively,
PLA2 was preincubated with HA or dHA for 30 min
in a 378C water bath, after which liposomes in Tris
buffer (100 mM, pH 8.0) supplemented with Ca2 +
(10 mM) were added to each of the mixtures to a ®nal
volume of 800 ml per mixture. The reactions were
allowed to continue for an additional 40 min in a
shaking water bath at 378C, after which they were
stopped by the addition of 3 ml of termination solution
consisting of chloroform : methanol : 2 N hydrochloric
acid at a ratio of 166 : 133 : 5 in the presence of 1 ml acid
saline. The lipids were extracted into an organic phase,
dried under a stream of nitrogen, resuspended in 40 ml
chloroform : methanol (1 : 1), chromatographed on silica
thin-layer plates and eluted in chloroform : methanol :
water (65 : 35 : 5) w27x. The band corresponding to the
NBD caproic acid (C6-NBD-FA) was scraped off and
extracted into chloroform : methanol (1 : 1). Its ¯uorescence intensity was measured by excitation at 470 nm
and emission at 535 nm w27x.
Interaction between HA and liposomes
The potassium bromide density test was applied to
determine whether a complex of HA with phospholipid
(HA±PL complex) is formed during incubation of the
lysosomes with HA. Samples of HA (2 ml, 5 mguml),
liposomes and their mixture were placed at the bottom
of three SW-41 centrifugation tubes and potassium
bromide gradients (1.005±1.21 guml) were layered on top
w28x. The tubes were then centrifuged at 40 000 r.p.m. for
21 h at 48C, after which they were examined for the
locations of HA, phospholipid and their mixture. HA
was detected by the use of the stain-all reagent, a blue
colour being formed by their interaction w29x. The liposomes were detected by C6-NBD-PC ¯uorescence.
The high-density HA band was expected to be located
at the bottom of the gradient, while the band of lowdensity liposomes was expected to be at the top of the
gradient. The nature of the interaction between HA and
338
D. W. Nitzan et al.
liposomes determines the location of the components
of the mixture in the gradient.
Results
PLA2 Activity
C6-FA (fluorescence units)
The inhibitory effect of HA on the hydrolysis of the
phospholipid substrate by exogenous PLA2 is shown
in Fig. 1. Dose-dependent inhibition of PLA2 activity
occurred in the presence of HA (®nal concentration
0±6.88 mguml). The rate of inhibition showed a decline:
a dramatic reduction in PLA2 activity (by 66%) occurred
in the presence of 1.22 mguml HA. Increasing concentration of HA was associated with a decreasing rate of
inhibition, to the point that PLA2 activity was almost
entirely blocked at physiological HA concentrations.
dHA (0.6±2.5 mguml) failed to inhibit hydrolysis of phospholipid by PLA2 (Fig. 2). In fact, there was a slight
degree of acceleration rather than inhibition in PLA2
activity in the presence of increasing concentrations of
dHA. It is known that HA, an antioxidant, may act as
a proactivator when degraded by free radicals, as is
frequently the case with oxidation products w30, 31x. The
same results were obtained (i) when HA or dHA was
incubated with liposomes to which PLA2 was later
added, and (ii) when HA or dHA was incubated with
PLA2 and phospholipid was added subsequently.
To investigate whether the inhibition of PLA2 activity is due to protection of the liposomes by HA, the
absorption of the latter to the lipid membrane was
determined with the potassium bromide density test. The
interaction between the two components, i.e. HA and
phospholipids, yielded a band in the gradient far below
the location of the low-density phospholipid alone, but
close to the high-density HA band, con®rming that an
HA±PL complex membrane had been created (Fig. 3).
The possibility that HA in the solution interacts with
PLA2 was also considered, but separation between the
HA and PLA2 using column chromatography was technically dif®cult because of the viscosity of HA. Inhibition consequent on interaction between the enzyme and
the free polymer in the solution was found to be negligible
compared with interaction with polymer adsorbed to
the membrane surface w32x.
Discussion
Hyaluronic acid (mg/ml)
PLA2 Activity
C6-FA (fluorescence units)
FIG. 1. Dose-dependent inhibition of PLA2 activity by increasing concentrations of HA.
In the last decade, HA has gradually lost its status as the
key component responsible for boundary lubrication.
However, the results of the present in vitro study suggest
that HA might have an indirect role in the boundary
lubrication of synovial joint. A major role in lubrication
has been assigned to SAPL w17±21x, which constantly
adapt themselves by means of remodelling, and within
the framework of this process PLA2 is probably responsible for the lysis of SAPL. When the existing situation
Hyaluronic acid (mg/ml)
FIG. 2. dHA accelerates rather than inhibits PLA2 activity.
FIG. 3. Potassium bromide density gradient. The liposomes
represented by the ¯uorescent band are located at a lowdensity level (left tube). The liposomeuHA mixture is located at
a high-density level, below the band of liposomes alone. This
con®rms that HA binds to the liposomes, forming a complex
(right tube).
Hyaluronic acid protects against PLA2 activity
becomes unbalanced and the lysogenic activity of this
enzyme becomes uncontrolled, there may be an actual
threat to the integrity of the SAPL. The steady-state
condition of the joint is indicative of the presence of an
ef®cient defence mechanism for the SAPL.
It was demonstrated in the present in vitro study that
HA interacts with liposomes, indicating that this lends it
the capability of protecting phospholipids against lysis
by exogenous PLA2. As may be inferred from the
FIG. 4. Synovial joint lubrication system and its partial
collapse. (a) SAPL cover the articular surfaces (arrows).
High molecular weight HA is attached to the SAPL as ¯uid
®lm. PLA2 is present in the joint. HA protects the SAPL from
attack by PLA2. (b) After excessive overloading of the joint,
free radicals may be produced, and thereby degrading the HA.
In its degraded form HA does not inhibit PLA2 activity, thus
enabling lysis of the SAPL layer. (c) On lysis of the SAPL, the
articular surfaces are stripped of their lubricants, exposing
them to unwanted effects. Because these surfaces are smooth
and possess high surface energy, friction is generated between
the `naked' articular surfaces (arrows). Such friction is probably the prime mover in the degenerative changes in the joint.
339
potassium bromide density test, HA adheres to the
liposomes ®rmly, thus forming a barrier against lysis by
PLA2. The degradation of HA by reactive oxidative
species impairs this function, thereby exposing the liposomes to lysis by PLA2. These in vitro results seem to
imply that HA plays an indirect role in joint lubrication
in functioning synovial joints by protecting the integrity
of SAPL (Fig. 4a)
It must be noted, however, that excessive joint loading
produces free radicals, which are capable of degrading
HA w26x. In its degraded form, HA is presumably unable
to inhibit PLA2 activity, and without such protection
SAPL become accessible to hydrolysis (Fig. 4b and c).
It is warranted to assume that, in these circumstances,
HA not only loses its role as a protector of phospholipids
but also increases their hydrolysis by PLA2. It is well
known that under certain circumstances antioxidants
may act w30, 31x as pro-oxidants (oxidation mediators),
accelerating the lysis of SAPL.
What is the importance of the integrity of lubrication
with regard to joint function? How does the elimination of SAPL affect joint function w10, 11, 33±35x? The
uncovered articular surfaces are elastic smooth planes
w36x with high surface energy w22, 37x; as such they
generate increased friction between the surfaces when
lacking in lubrication. Increased friction plays an
important role in a variety of joint disorders w12, 38±41;
D. W. Nitzan, submitted for publicationx, which may
all culminate in deteriorating degenerative joint disease.
In a similar fashion, the high molecular weight HA
inhibits angiogenesis, which probably accounts for the
avascular, white appearance of the healthy articular
surface w13x. The acceleration of angiogenesis by dHA,
on the other hand, explains the typical capillary-rich
appearance of the in¯amed articular surface w13x.
There is a comparable model of protection at the
cellular level that underscores our theory: PLA2 is
secreted into the extracellular ¯uid, where it constitutes
an active component in the in¯ammatory process, posing
a threat to the cell membrane phospholipids of the
target cells w42x. It has been shown w42x that a mechanism
similar to that described for SAPL in this study acts at
the cellular level: cell-surface proteoglycans protect the
cell membrane from being lysed by PLA2, a condition
that is abrogated in the presence of reactive oxidative
species w42x. The latter degrade cell-surface proteoglycans, rendering the membrane phospholipids accessible
to hydrolysis by PLA2.
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