Vol. 283, Issue 6, H2485-H2494, December 2002
Relationship between lymph and tissue hyaluronan in skin and
skeletal muscle
Shayn E.
Armstrong and
Donald R.
Bell
Center for Cardiovascular Sciences, Albany Medical College,
Albany, New York 12208-3479
 |
ABSTRACT |
The size of hyaluronan was compared
between tissue and lymph using a combination of agarose gel
electrophoresis and radiometric assay. Prenodal lymph was collected
from heel skin and the gastrocnemius muscle in anesthetized
rabbits. The major fraction of hyaluronan in both tissues had a
molecular weight >4 million. Lymph contained primarily
low-molecular-weight hyaluronan (<0.79 × 106),
which was absent from tissue. Volume loading produced a preferential increase in the flux of low-molecular-weight hyaluronan, indicating that tissue contains a small quantity of mobile, low-molecular-weight hyaluronan. The maximum daily removal of hyaluronan by lymph was <1%
of the tissue content. The amount of lysosomal hyaluronidase activity
in tissue was more than enough to account for a rapid turnover of
hyaluronan. The data support the conclusion that lymph drainage is not
significant in the normal catabolism of hyaluronan and may represent a
small amount that becomes detached from the pericellular and
extracellular matrixes.
extracellular matrix; hyaluronan catabolism; hyaluronidase
activity; molecular weight
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INTRODUCTION |
HYALURONAN IS A MAJOR
COMPONENT of pericellular and extracellular matrixes (19,
30). It is a linear, 
-1,4-linked polymer of the disaccharide
D-glucuronic acid
(1--3)-N-acetyl-D-glucosamine. The molecular
weight is considered polydisperse depending on the number of repeating
disaccharide units in the chain. It is synthesized at cell membranes as
a very large polymer with a molecular weight of ~6 × 106 (4, 12). Besides modulating tissue
hydration and transvascular fluid balance, it can stabilize the
extracellular matrix by binding to specific proteins called
hyaladherins. During tissue development or injury, hyaluronan helps
regulate cell motility, invasion, and proliferation by binding to cell
surface receptors and activating intracellular signaling pathways. Many
of these diverse functions depend on the size of the polymer.
Low-molecular-weight hyaluronan appears stimulatory for cell
proliferation and migration, whereas high-molecular-weight hyaluronan
is inhibitory (19). Although high-molecular-weight
hyaluronan inhibits angiogenesis, low-molecular-weight fragments induce
tube formation by cultured endothelial cells through binding to the
cell surface receptor CD44 (21, 28). These observations
suggest that both the size and amount are important determinants of
hyaluronan function within tissues even though the size has not been
well characterized.
Skin contains approximately one-half of the hyaluronan in the
body, and the turnover is rapid with a half-life of ~2 days (23, 29). Hyaluronan is specifically involved with the
migration of basal keratinocytes toward the outer cornified layer
during wound healing (20). Although the catabolism is not
well understood, the presence of hyaluronan in lymph leads to the
proposal that lymphatic drainage was an important catabolic pathway
(15, 30). On the basis of measurements of the hyaluronan
content in skin lymph and tissue and the rapid removal of
subcutaneously injected low-molecular-weight hyaluronan, Reed and
colleagues (8, 24) proposed that the extravascular space
contains two distinct pools of hyaluronan. One pool represented
recently synthesized hyaluronan attached to matrixes, whereas the other
pool represented hyaluronan released from the matrixes and drained by
the lymphatics. Increasing lymph hyaluronan flux would increase the
turnover of the free pool without influencing the bound pool. In a more
recent study (22), they questioned the significance of
this proposal because the removal rate of labeled high-molecular-weight
hyaluronan after a subcutaneous injection did not increase during a
condition known to increase lymph flux.
Our hypothesis was that the size distribution of hyaluronan in skin
would reflect the two pools of hyaluronan. The major fraction of
hyaluronan, corresponding to the matrix pool, would have a high
molecular weight. A smaller fraction in the tissue would have a lower
molecular weight and a size distribution similar to that found in
lymph. We further proposed that increasing lymph hyaluronan flux with
volume expansion would decrease the amount of low-molecular-weight
hyaluronan in the tissue without altering the amount of
high-molecular-weight hyaluronan. With the use of a combination of gel
electrophoresis and a radiometric assay, we measured the size of
hyaluronan in lymph and tissue to test this hypothesis. Volume
expansion was used to increase lymph hyaluronan flux. Besides skin, we
studied skeletal muscle because the much lower hyaluronan content and
the absence of keratinocytes may influence the relative size of the two pools.
We used anesthetized rabbits because lymph can be collected separately
from either skin or skeletal muscle of the hind leg. A difficulty in
comparing lymph hyaluronan flux with tissue hyaluronan content is
estimating the weight of tissue drained by the cannulated lymphatic. In
a previous study using rabbits (33), the weight of heel
skin drained by the cannulated lymphatic was estimated by comparing the
transvascular clearance of labeled albumin with the lymphatic clearance
of endogenous albumin. In the present study, we estimated the weight of
skeletal muscle drained by the cannulated lymphatic using the same
procedure. The values for the weight of skin or skeletal muscle drained
by the lymphatic was used to compare the lymph flux of hyaluronan with
the amount in the tissue.
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METHODS |
Animal preparation.
A detailed account of the surgical procedures has been previously
reported (18, 33). Protocols for animal use conformed with
the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996) and were approved by the Institutional Animal Care and Use Committee at Albany Medical College.
In brief, New Zealand White rabbits of either sex, weighing from 2.0 to
3.0 kg, were anesthetized with pentobarbital sodium (25-35 mg/kg)
and were given supplemental doses as required. Body temperature was
maintained at 39°C with a heating blanket and a Yellow Springs
Instruments rectal probe thermistor. After tracheotomy, a heparinized
cannula was placed into the right carotid artery for blood collections
and arterial blood pressure recordings using a Statham pressure
transducer connected to a Grass polygraph. Cannulas were placed in the
right external jugular vein for intravenous infusions.
For collection of skin lymph, the lymphatics located near the calcaneal
tributary vein of each leg were tied off, and a single, prenodal,
popliteal lymphatic was cannulated using polyethylene tubing heated and
pulled to the desired size. The collected lymph was from an area of
heel skin weighing 1.6 g dry wt (6 g wet wt) with negligible
contamination from other tissues (33). For collection of
skeletal muscle lymph, T1824-bound albumin was injected into the heel
skin of each leg to identify contamination from skin lymph. In each
leg, the lymphatics within the femoral sheath were ligated, and a
single lymphatic was cannulated distal to the inguinal node with
Silastic tubing (0.3 mm inner diameter and 0.64 mm outer diameter). The
initial lymph collected was blue from the T1824 injection. The
popliteal node was exposed, and the efferent lymph duct was ligated. If
lymph flow remained elevated or if the collected lymph did not become
clear after 1-2 h, data collection from that leg was terminated.
The procedure collects lymph predominately from the gastrocnemius and
soleus muscles (18), although the weight of tissue has not
been determined.
Lymph flow (in
ml · h
1 · g
dry wt1) was calculated by dividing the sample volume by
the collection time and the weight of tissue drained by the cannulated
lymphatic. Lymph was collected for 30-min intervals in weighed,
heparinized vials. Sample volume was determined using weight and
assuming that the density of lymph was 1. For both preparations, lymph
collection was done with the rabbit lying prone, and the legs were
moved passively at 60 times/min to promote lymph flow. A heparinized
sample of blood (0.8 ml) was collected at the midpoint of each lymph collection.
The concentration of hyaluronan in lymph was measured using a
radiometric assay. The concentration of hyaluronan with a molecular weight >0.79 × 106 or 4 × 106 was
measured using agarose gel electrophoresis. The concentration of
low-molecular-weight hyaluronan (<0.79 × 106) was
calculated as the concentration of hyaluronan, measured using the
radiometric assay, minus the concentration of hyaluronan with a
molecular weight >0.79 × 106. The concentration of
intermediately sized hyaluronan was calculated as the concentration of
hyaluronan with a molecular weight >0.79 × 106 minus
the concentration of hyaluronan with a molecular weight >4 × 106. The lymphatic flux for each species was calculated by
multiplying the lymph flow by the respective lymph concentration.
Lymphatic flux of hyaluronan was increased by volume expanding the
animals with lactated Ringer solution (Baxter). An intravenous infusion
of 150 ml/kg body wt was given at a rate of 1.3 ml · min1 · kg
body wt1. The duration of the infusion was 2 h.
After the infusion was ended, lymph and plasma samples were taken for
an additional 2 h. The animal was killed by an overdose of
pentobarbital sodium, and either the heel skin or gastrocnemius muscle
was removed. Separate animals that did not receive an infusion were
controls. After the tissue was removed, a section was weighed and dried to a stable weight at 60°C to calculate the wet weight-to-dry weight
ratio. The rest of the tissue was frozen at 
80°C for later analysis. Although the heel skin was shaved with electric clippers at
the beginning of the experiment, additional hair was removed from the
dried skin sample with a razor, and the weight was corrected for the
residual hair.
Tissue digestion and radiometric assay.
Frozen sections of tissue (300-350 mg) were weighed and placed in
2 ml of 0.15 M Tris, 0.15 M NaCl, 0.01 M CaCl2, and 5 mM deferoxamine mesylate, pH 8.3, containing 40 units of protease (Sigma)
for 8 h at 55°C. Deferoxamine was included to inhibit any
hyaluronan depolymerization by iron released during the digestion procedure (1). After incubation, the samples were
centrifuged at 53,000 g for 15 min at 4°C, and the
supernate was collected for analysis. For the radiometric assay, an
aliquot of the tissue digest was placed in a boiling water bath for 20 min to inactivate the protease. Digestion buffer containing protease
and no hyaluronan was used as a blank. The influence of the protease on
the binding of labeled hyaluronan binding proteins (HABP) to the
hyaluronan-Sepharose was always <5% after boiling. For both assays, a
3-fold dilution for skeletal muscle and a 12-fold dilution for skin was
made using 0.15 M Tris, 0.15 M NaCl, 0.01 M CaCl2, and 5.0 mM deferoxamine mesylate; pH 8.3.
The tissue content of hyaluronan irrespective of size, hyaluronan with
a molecular weight >0.79 × 106, and hyaluronan with
a molecular weight >4 × 106 was calculated. The
tissue content (µg/g wet tissue wt) was calculated by multiplying the
digest concentration by the volume of fluid present during digestion
and dividing by the weight of the tissue sample. These values were
converted to units of micrograms per gram dry weight by multiplying by
the wet weight-to-dry weight ratio.
The concentration of hyaluronan irrespective of size was measured using
a radiometric assay as described previously (1, 9).
Hyaluronan from Streptococcus (Calbiochem) was used as a
standard. Procedures for the isolation of HABP from cartilage and the
preparation of hyaluronan-conjugated Sepharose were described previously (1, 32). HABP were labeled with
125I (125I-HABP) using the lactoperoxidase
method (14). To protect the binding site, 300 µg HABP
was incubated with 225 µg rooster comb hyaluronan in 0.05 M phosphate
buffer, pH 7.5, before labeling with 2 mCi (74 MBq) 125I.
The labeled HABP were dialyzed (25,000 mol wt cutoff) against 4 M
guanidine HCl and applied to a Sephacryl S-400 gel filtration column
equilibrated in 4 M guanidine HCl to separate labeled HABP from
hyaluronan. Fractions containing the peak activity were mixed with 10 mg BSA, concentrated using an Amicon Ultrafiltration unit with a YM10
membrane, and stored at 4°C. The specific activity was between 25 and
45 kBq/µg.
Agarose gel electrophoresis.
The concentration of hyaluronan with determined molecular weight
characteristics was measured in samples of lymph and tissue digest
using gel electrophoresis as previously described (1, 10).
A 0.5% agarose (GIBCO-BRL) gel was made in Tris-sodium acetate-sodium
EDTA (TAE) buffer (40 mM Tris, 5 mM sodium acetate, and 0.8 mM sodium
EDTA; pH 7.9). To each lane, 10 µl lymph, diluted tissue digest, or a
standard was applied. The samples were electrophoresed at 50 V for
3 h in TAE buffer using a DNA Plus Horizontal Gel system from USA
Scientific. Hyaluronan was transferred from the gel to Hybond
H+ (Amersham Pharmacia) membranes using upward capillary
blotting. Optimum transfer required the overnight passage of 550 ml TAE buffer into a stack of blotting pads that was 6 cm thick. After transfer, membranes were blocked by incubation in 0.1 M sodium acetate-1 M NaCl containing 0.05% Tween 20, 2% nonfat dry milk, and
0.1% sodium azide, pH 6.0, for 6 h at 37°C. Membranes were subsequently transferred to 20 ml fresh solution containing 400 kBq
125I-HABP and incubated overnight at 4°C. After being
washed, X-ray film was exposed to the membrane for 4-6 h at
80°C and developed. A Bio-Rad GS-700 Imaging Densitometer and
Molecular Analyst software were used for quantitative analysis of the autoradiograph.
Both molecular weight and concentration standards were applied to each
gel. A series of hyaluronan standards with defined molecular weights
was generously donated by Dr. O. Wik (Pharmacia; Uppsala, Sweden). The
weight-averaged molecular weights of these standards were 0.20, 0.48, 0.79, 1.2, 2.0, 3.9, and 5.0 × 106. Each standard was
applied to different lanes. A calibration curve for molecular weight
was constructed from the autoradiograph. For each lane containing a
standard, the mobility of the peak (maximum lane intensity) was plotted
against the logarithm of the peak molecular weight and a straight line
fit using least squares. To verify the molecular weight corresponding
to the peak, the standards were chromatographed on a Sephacryl S-1000
column (1.6 × 97 cm). The column was calibrated using the
weight-averaged molecular weight as described by Laurent and Granath
(7). For each standard, the molecular weight corresponding
to the peak in the chromatogram was estimated from the standard curve
and used for the molecular weight of the peak in the electrophoretic profiles on the autoradiograph.
We calculated the concentration of hyaluronan with a molecular weight
>0.79 × 106 or 4 × 106 in lymph
and tissue digests. Concentration measurements were made by making
serial dilutions of the 3.9 × 106 molecular weight
standard and determining the concentration using the radiometric assay.
Each dilution was applied to separate lanes of the gel. A standard
curve for concentration was constructed from the autoradiograph. For
each standard, the total lane intensity was plotted against its
concentration, and a straight line was fit using least squares. The
relationship was linear between 1 and 10 µg/ml (10-100 ng
applied amount). For lymph and tissue samples, the lane intensity on
the autoradiograph was measured after setting the length of the lane to
correspond to a molecular weight of 0.79 × 106 or
4 × 106, determined from the molecular weight
calibration. For each sample, the lane intensity was converted to
hyaluronan concentration using the standard curve. Because the
concentration standards used in gel electrophoresis were calibrated
with the radiometric assay, the measurements from the two methods were
directly comparable.
Tissue hyaluronidase activity.
Hyaluronidase activity was measured in the liver and skin by
determining the initial velocity of end-product production as described
previously (2, 3). Tissue (0.5 g) was homogenized in 1.5 ml of 0.01 M sodium acetate and 0.15 M NaCl, pH 5.0, at 4°C using a
Brinkaman Polytron homogenizer. Hyaluronidase activity was measured in
the supernate after centrifugation at 53,000 g for 15 min.
Aliquots of tissue supernate (0.2 ml) were incubated with 0.4 ml of
0.15 M sodium acetate, 0.15 M NaCl, and 0.75 mg/ml rooster comb
hyaluronan, pH 4.0, for varying lengths of time. Isotonic saline
instead of tissue supernate was used as a blank. The reaction was
neutralized by the addition of 15 µl of 5 M NaOH. After
centrifugation, the concentration of N-acetyl-glucosamine at
the reducing end of hyaluronan was measured using the method of Reissig
et al. (25). The increase in the concentration was linear
with time up to 8 h, and the initial velocity was measured as the
slope during the first 6 h. Hyaluronidase activity was calculated
as the initial velocity multiplied by the dilution factor and volume of
tissue supernate and divided by tissue weight. Tissue weight was
converted to dry weight using the wet weight-to-dry weight ratio.
We used the diffusion plate assay described by Richman and Baer
(26) to measure the hyaluronidase activity in skeletal
muscle, plasma, and lymph due to the low activity present in these
samples. We assumed that the diffusion characteristics for the enzymes in the sample were the same as those for the enzymes in skin and used
serial dilutions of skin supernate as a standard. The procedure for
homogenization of skeletal muscle was the same as skin. A 1% agarose
gel containing 0.5 mg/ml rooster comb hyaluronan was made in 0.1 M
sodium acetate and 0.15 M NaCl (pH 4.0) buffer. Samples of 10 µl were
applied to wells in the gel and incubated for 18 h at 37°C. The
undigested hyaluronan was precipitated by placing the gel into 0.28 M
cetylpyridinium chloride overnight. The diameter of clear circles,
representing digested hyaluronan, was related to the logarithm of the
concentration of enzyme in the applied sample. Sample concentrations
were converted to activities by multiplying the concentration by the
activity measured in the skin supernate that was used as a standard.
Weight of skeletal muscle drained by lymph.
In a separate experiment, the weight of skeletal muscle drained by the
cannulated lymphatic was estimated by comparing the transvascular
clearance of labeled albumin with the lymphatic clearance of endogenous
albumin as we described previously for skin (33). A 1-h
infusion of labeled albumin was started after obtaining a stable lymph
flow. A bolus of 135 µCi (5 MBq) of 125I-labeled albumin
was injected intravenously followed by an infusion to maintain the
plasma concentration constant with time. Plasma samples were taken at
15 and 45 min after the beginning of the tracer infusion to be certain
that the plasma tracer activity was not changing with time. Three
minutes before the experiment was ended, 200 µCi (7 MBq) of
131I-labeled albumin were injected intravenously to measure
the plasma volume in tissue. After the 3-min mixing time, a final
plasma sample was taken, the animal was killed by an overdose of
pentobarbital sodium, and the gastrocnemius and soleus muscles were
removed. Samples of the muscles were weighed, counted for radioactivity with plasma and lymph samples, and dried to a stable weight at 60°C.
The wet weight-to-dry weight ratio was calculated as the fresh tissue
weight divided by the dry weight. The concentration of
non-protein-bound activity was determined in plasma using
ultrafiltration with an Amicon Centricon-30.
The concentration of endogenous albumin in lymph and plasma samples was
measured using rocket electroimmunoassay as described previously
(33). Antibody to rabbit albumin was purchased from Cappel
Laboratories, and purified rabbit albumin was used as a standard. As
described previously (18, 33), rabbit albumin was purified
using affinity and gel filtration chromatography. It was labeled with
125I or 131I using a modified chloramine-T
procedure. The labeled protein was placed in dialysis (Spectropore 6:
25,000 mol wt cutoff) for at least 5 days to reduce the fraction of
non-protein-bound activity below 0.3%.
For each tissue sample, the volume of plasma (VP) was
calculated as the amount of 131I in the tissue sample (in
disintegrations · min1 · g
wet wt1) divided by the plasma concentration (in
disintegrations · min1 · ml1).
The transvascular clearance of labeled albumin (CT) was
calculated using Eq. 1, where RWD is the wet
weight-to-dry weight ratio and T is the number of hours the
labeled protein was in the circulation
|
(1)
|
The weight drained by the cannulated lymphatic (W)
was calculated using Eq. 2. This equation provides a minimum
value for the weight because it assumes that transvascular transport
for albumin is by convection only (33). Lymph clearance of
endogenous albumin (CLEA) was calculated as lymph flow
times the lymph-to-plasma concentration ratio for endogenous albumin.
Lymph clearance of labeled albumin (CLLA) was calculated as
the total radioactivity in lymph divided by the plasma concentration
|
(2)
|
Statistics.
Data are presented as means ± SE. A one- or two-way ANOVA with
repeated measures and Scheffé's test were used to test for significance between groups. Significance was set at P < 0.05.
| |
RESULTS |
Weight of skeletal muscle drained by lymph.
Lymph was collected from seven legs in six rabbits to estimate the
weight of skeletal muscle drained by the cannulated lymphatic. After
ligation of the popliteal node, lymph was collected for 4 h before
the infusion of labeled albumin was started. Lymph flow and the lymph
clearance of endogenous albumin remained constant with time during the
last 2 h of the experiment. Values measured during the 1-h
infusion of labeled albumin are presented in Table 1. Plasma albumin was 32 ± 2 mg/ml.
The plasma concentration for the labeled albumin did not change with
time. The difference between the initial and final concentrations was
5 ± 4%. The amount of non-protein-bound activity in the final
plasma sample was 0.30 ± 0.08%. The specific activity in the
final lymph sample relative to plasma was 6 ± 1%. Values for the
transvascular clearance of labeled albumin were not different between
the gastrocnemius and soleus muscles and were averaged. The wet
weight-to-dry weight ratio was 4.3 ± 0.1, and the vascular volume
in the tissue samples was 31 ± 2 µl/g dry wt. The tissue weight
drained by the cannulated lymphatic was equivalent to 17 g wet wt.
Skin hyaluronan.
Electrophoresis of digested skin showed a single major peak that had a
molecular weight greater than our highest standard. Figure
1 shows the electrophoretic pattern for a
sample of digested skin and skin lymph. For the tissue digest, the
concentration of hyaluronan irrespective of size was 100 µg/ml. The
concentration of hyaluronan with a molecular weight >4 × 106 was 64 µg/ml. The concentration of hyaluronan with a
molecular weight >0.79 × 106 was 92 µg/ml. The
pattern for lymph showed a shift toward lower-molecular-weight hyaluronan and greater polydispersity. For the lymph sample, the concentration of hyaluronan irrespective of size was 7.7 µg/ml. The
concentration of hyaluronan with a molecular weight >4 × 106 was 1.7 µg/ml, which corresponds to only 22% of the
hyaluronan in lymph. The concentration of hyaluronan with a molecular
weight >0.79 × 106 was 5.0 µg/ml, so that 35% of
the hyaluronan in the lymph sample had a molecular weight <0.79 × 106. Control measurements were obtained from eight legs
in four rabbits that were not volume expanded. Lymph was collected for
4 h, during which lymph flow did not significantly change with
time. Mean arterial blood pressure was 79 ± 8 mmHg. Lymph flow
was 0.08 ± 0.01 ml · h1 · g
dry wt1 using a value of 1.6 g dry wt for the weight
of tissue drained by the cannulated lymphatic (33). The
lymph concentration of hyaluronan irrespective of size was 8.1 ± 0.5 µg/ml. Lymphatic flux of hyaluronan was 0.6 ± 0.1 µg · h1 · g
dry wt1. The fraction of hyaluronan with a molecular
weight >4 × 106 was only 21 ± 2%. The
fraction of low-molecular-weight hyaluronan (<0.79 × 106) was 31 ± 2%.
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Fig. 1.
Densitometric scan of an autoradiograph of skin tissue
digest (solid line) and skin lymph (dashed line) after electrophoresis
on 0.5% agarose gels. Radiolabeled hyaluronan binding proteins were
used to detect hyaluronan specifically after transfer to a nylon
membrane. Vertical lines show the mobility for the indicated molecular
weight, obtained from the calibration curve.
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As shown in Fig. 2, volume expansion
produced a sustained increase in skin lymph flow and hyaluronan flux.
Measurements were obtained from seven legs in four volume-expanded
rabbits. Mean arterial blood pressure did not change during the
experiment and was not different from the control group. The baseline
values for lymph flow and lymphatic flux of hyaluronan were not
significantly different from the values in the control group. Lymph
flow increased threefold from baseline during the volume expansion and
remained elevated for the 2 h after the infusion was ended.
Lymphatic flux of hyaluronan increased to a peak value of 5.4 times the
baseline value during the infusion. It decreased from the peak to a
value 3.6 times the baseline value by the end of the experiment when tissue samples were taken. The lymphatic flux of hyaluronan with a
molecular weight >0.79 × 106 was three times the
baseline value both during and after the infusion. A smaller increase
was observed for hyaluronan with a molecular weight >4 × 106.
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Fig. 2.
Skin lymphatic flux of hyaluronan (A) and
lymph flow (B) with volume expansion. Values are plotted
against time after the start of the intravenous infusion of lactated
Ringer solution. Time represents the midpoint of a 0.5-h collection. B,
baseline; solid bar, duration of the infusion. In A, closed
circles represent hyaluronan measured with the radiometric assay,
closed squares represent hyaluronan with a molecular weight >0.79 × 106, and closed triangles represent hyaluronan with a
molecular weight >4 × 106. Values are means ± SE; n = 7. * P < 0.01 compared with
baseline from 0.25 to 3.75 h;  P < 0.05 compared with the final (3.75 h) value.
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The increase for low-molecular-weight hyaluronan was significantly
greater than that for high-molecular-weight hyaluronan. Figure
3 shows lymphatic fluxes of differently
sized hyaluronan from skin. Baseline values were not different from the
control values. The lymphatic flux of low molecular weight hyaluronan increased fivefold. The lymphatic flux of high-molecular-weight hyaluronan was only 2.7 times baseline.
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Fig. 3.
Skin lymphatic flux of differently sized hyaluronan.
Solid bars, hyaluronan with a molecular weight <0.79 × 106; open bars, hyaluronan with molecular weights between
0.79 × 106 and 4 × 106; shaded
bars, hyaluronan with a molecular weight >4 × 106.
Values are means ± SE; n = 8 separate control
animals and 7 animals for the baseline and final (3.75 h) values from
the experimental group. * P < 0.05 compared with
baseline.
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In contrast to lymph, skin did not contain a significant quantity of
low-molecular-weight hyaluronan. Figure 4
shows a summary of the content of hyaluronan in heel skin. The values
from the expanded animals were not different from the control values.
Measurements of hyaluronan with a molecular weight >0.79 × 106, using gel electrophoresis, were not different from
those using the radiometric assay. This observation suggests the
absence of a significant quantity of low-molecular-weight hyaluronan in
the tissue. The fraction of hyaluronan with a molecular weight >4 × 106 was 57 ± 4% and 57 ± 8% for control
and volume-expanded animals, respectively. These fractions were
significantly greater than those found in lymph. The wet weight-to-dry
weight ratio was 3.78 ± 0.05 for the control group. The value for
the volume-expanded group was 4.26 ± 0.07 and significantly
larger than control.
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Fig. 4.
Summary of hyaluronan content in heel skin. Solid bars,
hyaluronan content measured using the radiometric assay; open bars,
hyaluronan with a molecular weight >0.79 × 106;
shaded bars, hyaluronan with a molecular weight >4 × 106. Values are means ± SE; n = 8 control and 7 volume-expanded animals. * P < 0.01 compared with content measured with the radiometric assay.
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Skeletal muscle hyaluronan.
Control measurements for skeletal muscle were obtained from seven legs
in seven rabbits that were not volume expanded. Lymph was collected for
4 h after ligation of the popliteal node. Measurements of
hyaluronan in lymph were made on the last 30-min collection just before
the tissue samples were taken. Mean arterial blood pressure was 90 ± 9 mmHg. Lymph flow was 0.012 ± 0.002 ml · h1 · g
dry wt1 using a value of 5.2 g dry wt for the weight
of tissue drained by the cannulated lymphatic (Table 1). The lymph
concentration of hyaluronan irrespective of size was 4.1 ± 0.6 µg/ml and was significantly less than that in skin lymph. Lymphatic
flux of hyaluronan was 0.050 ± 0.008 µg · h1 · g
dry wt1. The lymph from skeletal muscle contained a
higher fraction of low-molecular-weight hyaluronan than lymph from
skin. The fraction of low- and high-molecular-weight hyaluronan was
54 ± 6% and 7 ± 2%, respectively.
Measurements after volume expansion were obtained from five legs in
five separate rabbits. Mean arterial blood pressure did not change
during the experiment and was not different from the control group. We
did not obtain reliable baseline measurements due to the time required
to remove the residual skin lymph from the lymphatics after ligation of
the popliteal node. The measurements after volume expansion were
compared with the separate control group. Figure
5 presents a summary of the lymphatic
flux of hyaluronan from skeletal muscle. Lymph flow was 0.047 ± 0.006 ml · h1 · g
dry wt1, which was significantly greater than the control
value. Hyaluronan flux after expansion was five times the control
value. Similar to skin, volume expansion produced a preferential
increase in the lymphatic flux of low-molecular-weight
hyaluronan. The flux of low-molecular-weight hyaluronan was 0.22 ± 0.03 µg · h1 · g
dry wt1 and eight times the control value. The flux of
high-molecular-weight hyaluronan was not significantly different from
the control.
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Fig. 5.
Skeletal muscle lymphatic flux of hyaluronan. Solid bars,
hyaluronan measured using the radiometric assay; open bars, hyaluronan
with a molecular weight >0.79 × 106; shaded bars,
hyaluronan with a molecular weight >4 × 106. Values
are means ± SE; n = 7 control and 5 volume-expanded animals. * P < 0.01 compared with
control.
|
|
Although skeletal muscle contained much less hyaluronan than skin, the
electrophoretic pattern was very similar to that shown for skin tissue
in Fig. 1. Figure 6 shows a summary of
hyaluronan content in the gastrocnemius muscle. The values from the
volume-expanded animals were not different from the corresponding
values from the control animals. The amount of hyaluronan measured with
the radiometric assay was not different from the amount of hyaluronan with a molecular weight >0.79 × 106 measured using
gel electrophoresis. The fraction of hyaluronan with a molecular weight
>4 × 106 was 58 ± 3% and was not
significantly different from the value for skin. The wet weight-to-dry
weight ratio for the control was 3.95 ± 0.09. The value for the
expanded group was 4.29 ± 0.06 and was significantly greater than
the control.
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Fig. 6.
Summary of hyaluronan content in gastrocnemius muscle.
Solid bars, hyaluronan content measured using the radiometric assay;
open bars, hyaluronan with a molecular weight >0.79 × 106; shaded bars, hyaluronan with a molecular weight
>4 × 106. Values are means ± SE;
n = 7 control and 5 volume-expanded animals.
* P < 0.01 compared with content measured with the
radiometric assay.
|
|
Hyaluronidase activity in control tissue.
The hyaluronidase activity at pH 4.0 was determined in tissues taken
from control rabbits. As shown in Fig. 7,
the activity in skin was 40% of the activity in the liver and more
than six times the activity in skeletal muscle. The activity in plasma and lymph was measured from eight control rabbits. The activity in
plasma was 0.14 ± 0.03 µmol N-acetyl-glucosamine
produced · h1 · ml1.
The activity in skin lymph was 0.04 ± 0.01 µmol
N-acetyl-glucosamine produced · h1 · ml1
and was significantly less than the value for plasma.
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|
Fig. 7.
Hyaluronidase activity at pH 4.0 in the liver
(n = 5), heel skin (n = 8), and
gastrocnemius muscle (n = 7) from control rabbits.
Activity represents the amount of N-acetyl-glucosamine at
the reducing end of hyaluronan produced per hour. Values are means ± SE. * P < 0.05 compared with the liver;
P < 0.05 compared with skin.
|
|
| |
DISCUSSION |
The size of hyaluronan in lymph was considerably smaller than the
size in the tissue. Increasing lymph flow with volume expansion produced a preferential increase in the lymphatic flux of
low-molecular-weight hyaluronan. Because lymph was collected from
prenodal lymphatics, the low-molecular-weight hyaluronan was not
produced by degradation within lymph nodes. These results support in
part our hypothesis that tissue contains two pools of hyaluronan with
different sizes. Although the tissue measurements cannot distinguish
between matrix associated and disassociated hyaluronan, the movement
into lymph may be used to characterize a mobile pool within the tissue.
This pool was predominately low-molecular-weight hyaluronan.
Measurements in tissue did not show a significant amount of
low-molecular-weight hyaluronan, as we originally proposed. The amount
of low-molecular-weight hyaluronan in tissue may have been too small to
be measured. The size of hyaluronan in tissue and lymph did not appear
related to specific functions in skin because the results from skin and skeletal muscle were qualitatively similar.
We studied the size of hyaluronan using a combination of agarose gel
electrophoresis and a radiometric assay. Both methods used radiolabeled
HABP that were isolated from cartilage and specific for hyaluronan.
Because hyaluronan has one negative charge on each disaccharide, the
electrophoretic mobility depended on the number of repeating
disaccharides in the linear chain (10). Both concentration
and molecular weight standards were applied to the agarose gel. The
standards for concentration were purified high-molecular-weight
hyaluronan. The concentration was measured with the radiometric assay
so that measurements from the two methods could be directly compared.
The concentration of hyaluronan with a molecular weight >0.79 × 106 or 4 × 106 was measured in lymph or
tissue digest using electrophoresis. The concentration of hyaluronan
irrespective of size was measured using the radiometric assay. This
assay measures a decasaccharide (2 kDa) or larger (1). The
use of both assays avoids the problems associated with measuring
low-molecular-weight hyaluronan using agarose gel electrophoresis
(1). The concentration of low-molecular-weight hyaluronan
was calculated as the difference between the concentration measured
with the radiometric assay and the concentration of hyaluronan with a
molecular weight >0.79 × 106.
We found no evidence for low-molecular-weight hyaluronan in tissue from
either skin or skeletal muscle. Values for the amount of hyaluronan
with a molecular weight >0.79 × 106 were not
different from the values measured using the radiometric assay. The
absence of a significant amount of low-molecular-weight fragments in
tissue was consistent with previous studies on noncancerous tissue
showing no extracellular hyaluronidase activity at physiological pH
(15). Our values for the hyaluronan content measured using the radiometric assay agreed with values reported previously for hind
leg skin and skeletal muscle from the rat (34). Despite the large difference in hyaluronan content between skin and skeletal muscle, the electrophoretic patterns were similar. The pattern showed a
single high-molecular-weight peak for both tissues. Lee and Cowman
(10) obtained a very similar pattern for human synovial fluid using agarose gel electrophoresis and staining with Stains-All. They estimate that the peak for synovial fluid corresponded to a
molecular weight of 6 × 106 using a cross-linked
derivative of hyaluronan as a high-molecular-weight standard. They
emphasized that this is an approximation because cross-linked
hyaluronan may not behave exactly like the linear polymer. The
similarity between our electrophoretic patterns and theirs suggests
that the peak for skin and skeletal muscle corresponds to a molecular
weight of ~6 × 106, similar to synovial fluid.
The fraction of hyaluronan with a molecular weight >4 × 106 was at least 57% for both skin and skeletal muscle. A
similar value was found for synovial fluid (10). The
similar fractions for high-molecular-weight hyaluronan among tissues
may indicate a common synthetic pathway. Hyaluronan synthase, which has
three isoenzymes, is located in the cell membrane of many cells
(4). Li and Heldin (12) measured the size of
hyaluronan produced by mesothelioma cells transfected with hyaluronan
synthase-2 using gel filtration chromatography. Their chromatographic
pattern was very similar to the electrophoretic pattern for skin tissue
shown in Fig. 1. Similar to our results, the majority of hyaluronan had
a molecular weight >4 × 106. The size of hyaluronan
in skin and skeletal muscle may reflect the high-molecular-weight
product of hyaluronan synthase with little extracellular
depolymerization. Thus the molecular weight of hyaluronan in tissue may
not be as polydisperse as generally proposed.
Previous reports on the size of hyaluronan in skin (17,
23) showed a much lower molecular weight. In contrast to
synovial fluid, skin, or other solid tissues must be digested to
release hyaluronan associated with the extracellular or pericellular
matrix. Reed and colleagues (23) suggested that the
digestion procedure may cause depolymerization. Although tissue
hyaluronidase is not active at the pH used for digestion
(5), the release of iron from cells during the process may
cause depolymerization. Iron causes hyaluronan depolymerization by free
radical attack of the glycosidic linkages that is inhibited by
deferoxamine (13, 27). We found in preliminary experiments
that omitting deferoxamine from the digestion buffer resulted in a
time-dependent decrease in the molecular weight of hyaluronan. This
decrease was consistent with the earlier observations by Reed and
colleagues (23) and showed the need to include
deferoxamine in the digestion buffer.
Lymph contained a much smaller fraction of high-molecular-weight
hyaluronan than tissue. Only 7% of the hyaluronan in skeletal muscle
lymph had a molecular weight >4 × 106. Presumably,
the high-molecular-weight hyaluronan in tissue was associated with
pericellular and extracellular matrixes and could not easily move into
lymphatics. Lymph, however, contained a significant fraction of
low-molecular-weight hyaluronan that was not detected in tissue. The
fraction of hyaluronan with a molecular weight <0.79 × 106 was 31% and 54% in lymph from skin and skeletal
muscle, respectively. Further evidence for a small pool of dissociated
low-molecular-weight hyaluronan within these tissues was shown when
lymph flow was increased. Volume expansion caused an increase in lymph
flow and lymphatic flux of hyaluronan similar to a previous observation on the dog hind leg (24). There was little or no increase
in the flux of high-molecular-weight hyaluronan, whereas the flux of
low-molecular-weight hyaluronan was five times control for skin and
eight times control for skeletal muscle. These changes suggest that
low-molecular-weight hyaluronan within tissue is more mobile than
high-molecular-weight hyaluronan and can move into lymph. Although more
complex mechanisms may be involved, the increase in lymphatic flux of
hyaluronan after volume expansion may be due to a simple washout of
low-molecular-weight hyaluronan that is not associated with matrixes.
The amount of low-molecular-weight hyaluronan, however, appears to be
small because we could not measure it in tissue.
Volume expansion did not produce a significant decrease in tissue
hyaluronan content. The increases in lymphatic flux were small compared
with the hyaluronan content in the tissue. For skin, the increase in
flux (expanded minus baseline) was equivalent to 17 µg/g dry wt
during the 4 h of expansion. This value represented 0.7% of the
total hyaluronan content or 1.5% of the hyaluronan with a molecular
weight <4 × 106. Any decrease in tissue hyaluronan
due to this increased lymphatic flux would be much smaller than the
variation between animals or the error in the measurements. The
calculations and conclusion for skeletal muscle were similar to skin.
The absence of a change in tissue content was consistent with lymph
draining only a small fraction of the total amount of hyaluronan in tissue.
The lymphatic fluxes of hyaluronan may be overestimated due to the
method used to measure the weight of tissue drained by the lymphatic.
The method compared the transvascular clearance of labeled albumin to
the lymphatic clearance of endogenous albumin. Skin measurements have
been reported previously (33), and the measurements for
skeletal muscle were presented in Table 1. The values for both tissues
are within the ranges estimated using local dye injections
(18). The major assumption used in the method was that the
transvascular clearance of labeled albumin, corrected for the loss in
lymph, was equal to the lymphatic clearance of endogenous albumin. The
transvascular clearance represented a unidirectional flux due to the
negligible accumulation of labeled albumin in the extravascular space.
The lymphatic flux represented a net flux because there may be
diffusive movement across the microvascular wall from the extravascular
into vascular space. The extent of this movement is not known and
depends in part on the importance of convective transport across the
microvascular wall (16). The calculated tissue weight
represented a minimum weight because the unidirectional flux for
labeled albumin may have been greater than the net flux for endogenous
albumin. Thus the fraction of tissue hyaluronan removed by lymph was
probably overestimated in the present study.
Although the catabolism is not well understood, hyaluronan can be
removed from the extracellular space through lymphatic drainage or
through endocytosis followed by lysosomal degradation. Our data showed
that the daily removal of hyaluronan by lymph was small for both skin
and skeletal muscle. The lymphatic flux from skin was 0.6 µg · h1 · g
dry wt1, equivalent to 14 µg · 24 h1 · g dry wt1. This
value represented <0.6% of the tissue content. For skeletal muscle,
the daily flux was <0.4% of the tissue content using the control
value for lymphatic flux of 0.05 µg · h1 · g
dry wt1. Studies using radiolabeled acetate as a
precursor showed that the turnover of hyaluronan in rabbit skin was
rapid with a half-life of ~2 days (29). These
observations suggest that lymphatic removal represents <3% of the
catabolic pathway. This value represents a maximum because our
lymphatic fluxes may be overestimated. Studies using hyaluronan
conjugated to cellobiose showed that less than one-half of the
conjugate was removed through endocytosis, whereas the remaining
fraction was removed by lymph (8). These studies may have
overestimated the importance of lymph due to the use of a
low-molecular-weight conjugate. The small size may have permitted rapid
diffusion into the lymphatic system without substantial contact with
cellular receptors.
Because lymphatic flux of hyaluronan was too small to explain the rapid
turnover of hyaluronan in skin and skeletal muscle, we measured the
hyaluronidase activity at an acidic pH. This activity represented
lysosomal activity. Our value for the liver, equivalent to 0.9 µmol · h1 · g
wet wt1, was similar to the value of 0.8 µmol · h1 · g
wet wt1 reported for the rat liver (2). Our
value for skin was higher than the value Cashman and colleagues
(3) reported for partially purified extracts from rat
skin. They used a longer time to measure the production of
N-acetyl-glucosamine at the reducing end of hyaluronan. Loss
of activity during purification and the longer time may have
underestimated the activity. The hyaluronidase activity in skin and
skeletal muscle was 1.55 and 0.23 µmol
N-acetyl-glucosamine produced · h1 · g
dry wt1, respectively. This rate was equivalent to the
hydrolysis rate of -1,4 bonds. With the use of 419 g/mol for the
molecular weight of a disaccharide, the minimum amount of hyaluronan
that could be degraded was 0.65 and 0.10 mg · h1 · g
dry wt1 for skin and skeletal muscle, respectively. These
values are minimum estimates because the calculation assumed the
maximal number of bonds hydrolyzed for a single hyaluronan chain. These rates were much greater than lymph flux and more than enough to account for a rapid turnover of hyaluronan in these tissues. Thus the
limiting step in hyaluronan catabolism appears to be the rate of endocytosis.
Although the mechanism for internalization is not known, it may involve
a small amount of fragmentation at the cell surface. These fragments
would be released into the extracellular space and represent the small,
mobile pool of low-molecular-weight hyaluronan that appears in lymph.
Receptor-mediated endocytosis of hyaluronan has been studied in a
variety of cultured systems (30, 31). In contrast to liver
or lymphatic endothelial cells, internalization by keratinocytes or
fibroblasts appears primarily through the CD44 receptor. It is not
associated with clatherin-coated vesicles, caveolae, or pinocytosis.
Tammi and colleagues (30, 31) showed that cultured
keratinocytes internalized newly synthesized hyaluronan, which
presumably was high-molecular-weight hyaluronan. They found, however,
that most of the intracellular hyaluronan was located in early
endosomes and had a low molecular weight, suggesting partial
degradation before entering lysosomes. Hyal2, a type 1 hyaluronidase,
has been found associated with cell membranes (11). Although the optimal activity is at a low pH, changes in the cell membrane during endocytosis may enhance the activity, producing a
small amount of hyaluronan fragments. Irrespective of the mechanism that produces the low-molecular-weight fragments found in lymph, our
data shows that the amount of low-molecular-weight hyaluronan in either
lymph or tissue is very small compared with the amount of
high-molecular-weight hyaluronan in tissue.
In summary, both skin and skeletal muscle contain predominately
high-molecular-weight hyaluronan that was not removed by lymph after
expansion. Lymph contained mostly low-molecular-weight hyaluronan, indicating that tissue had a pool of mobile low-molecular-weight hyaluronan. The size of this pool was small because it could not be
measured in the tissue. The maximum daily removal of hyaluronan by
lymph was <1% of the tissue content, indicating that lymphatic drainage is not significant in the normal catabolism of hyaluronan. The
amount of lysosomal hyaluronidase activity in the tissues was more than
enough to account for the rapid turnover of hyaluronan.
| |
ACKNOWLEDGEMENTS |
We are grateful to Nina C. DeCocco and Bles Nuqui for excellent
technical assistance.
| |
FOOTNOTES |
This work was supported by National Institutes of Health Grant
AR-42445. S. E. Armstrong was supported by NIH Predoctoral Training Grant T32 HL-07194.
Address for reprint requests and other correspondence:
D. R. Bell, Center for Cardiovascular Sciences, MC-8, Albany
Medical College, Albany, NY 12208-3479 (E-mail:
belld{at}mail.amc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 1, 2002;10.1152/ajpheart.00385.2002
Received 2 May 2002; accepted in final form 16 July 2002.
| |
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ABSTRACT
INTRODUCTION
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