INVITED REVIEW
Structural determinants of antiproliferative activity of
heparin on pulmonary artery smooth muscle cells
Hari G.
Garg,
B. Taylor
Thompson, and
Charles A.
Hales
Pulmonary/Critical Care Unit, Department of Medicine, Massachusetts
General Hospital and Harvard Medical School, Boston, Massachusetts
02114
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ABSTRACT |
In addition to its
anticoagulant properties, heparin (HP), a complex polysaccharide
covalently linked to a protein core, inhibits proliferation of several
cell types including pulmonary artery smooth muscle cells (PASMCs).
Commercial lots of HP exhibit varying degrees of antiproliferative
activity on PASMCs that may due to structural differences in the lots.
Fractionation of a potent antiproliferative HP preparation into high
and low molecular weight components does not alter the
antiproliferative effect on PASMCs, suggesting that the size of HP is
not the major determinant of this biological activity. The protein core
of HP obtained by cleaving the carbohydrate-protein linkage has no
growth inhibition on PASMCs, demonstrating that the antiproliferative
activity resides in the glycosaminoglycan component. Basic sugar
residues of glucosamine can be replaced with another basic sugar, i.e.,
galactosamine, without affecting growth inhibition of PASMCs.
N-sulfonate groups on these sugar residues of HP are not
essential for growth inhibition. However, O-sulfonate groups
on both sugar residues are essential for the antiproliferative activity
on PASMCs. In whole HP, in contrast to an earlier finding based on a
synthetic pentasaccharide of HP, 3-O-sulfonation is not critical for
the antiproliferative activity against PASMCs. The amounts and
distribution of sulfonate groups on both sugar residues of the
glycosaminoglycan chain are the major determinant of antiproliferative activity.
heparitinase I; heparitinase II; glycosaminoglycan; heparan
sulfate; core protein;
-disaccharide; anticoagulation activity; biosynthesis
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INTRODUCTION |
HEPARIN (HP) was
discovered nearly 80 years ago and is presently used as an
anticoagulant drug (60). Besides its anticoagulant activity, HP has a variety of other biological and biochemical activities that include the following: 1) regulation of
lipid metabolism (17), 2) control of blood
fluidity at the endothelial surface (50), 3)
control of cell attachment to various proteins in the extracellular
matrix (ECM) (42, 43, 73), 4) binding with
acidic and basic fibroblast growth factors (3, 61), 5) binding to interleukin-3 and granulocyte-macrophage
colony-stimulating factor (27, 59), and 6)
inhibition of serotonin-induced pulmonary artery (PA) smooth muscle
cell (SMC) hypertrophy (44). HP stimulates endothelial
cell growth (69), whereas it inhibits the proliferation of
renal mesangial cells (65), rat cervical epithelial cells (1), transformed cell lines (5, 16, 30), and
systemic SMCs and PASMCs (18, 68). Of the types of
biological activities just mentioned, anticoagulation has been
extensively discussed in several reviews (6, 15, 36, 48,
76). Other biological activities
(2-5) have recently been reviewed (4,
21, 31, 57, 70). Regulation of vascular SMC proliferation by HP
was reviewed in 1989 (74) and 1994 (40).
These reviews specifically discussed which domain of HP is responsible
for the antiproliferative activity on aortic vascular SMCs. Briefly,
they suggested that 1) the anticoagulant and
antiproliferative properties of HP reside in different HP domains,
2) 3-O-sulfate on the internal glucosamine residue of a chemically synthesized pentasaccharide (Fig.
1) (63) is critical for the
growth-inhibitory capacity of the pentasaccharide (11),
3) the dodecasaccharide of HP contains the full
antiproliferative activity (10), 4)
2-O-sulfonation of glucuronic acid in HP is not essential for
antiproliferative activity (75), 5) the
relationship between the degree of N-desulfonation and the inhibition
of cell proliferation is not straightforward (71),
6) acetylation of the N positions of the N-desulfonated
glucosamine residues does not seem to restore the antiproliferative
activity (71), and 7) both O-sulfonation and
N-sulfonation are important for antiproliferative activity
(71).

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Fig. 1.
Antiproliferative pentasaccharide demonstrating the structure
critical for growth inhibition. Sequence of sugar residues
(left to right):
D-glucosamine-D-glucuronic
acid-D-glucosamine-L-iduronic
acid-D-glucosamine. *, Substituent essential for
growth inhibitory capacity of the pentasaccharide. For numbering of
sugar rings, see Fig. 3. [Reprinted from Garg et al.
(25). Copyright 1996, Academic Press.]
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Because vascular remodeling with SMC hypertrophy and hyperplasia
contributes to the high pulmonary vascular resistance seen in primary
as well as secondary pulmonary hypertension, interest continues in HP
as a possible therapeutic agent to reverse vascular remodeling. In
recent years, efforts have been made to establish which domain of the
HP polysaccharide is related to the inhibition of growth of PASMCs.
Several studies employing the following strategies, mild chemical
modification, fractionation, or enzymatic degradation, have appeared.
This review provides an update on the effects of HP and its fragments
on the inhibition of growth of PASMCs.
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HEPARIN STRUCTURE |
Arrangement of sugars in glycosaminoglycan chains.
HP is one of the members of the class of glycosaminoglycans
(GAGs; Table 1) (26) and
consists of alternating residues of a uronic acid (either
-D-glucuronic acid or
-L-iduronic acid) with a hexosamine (
-D-glucosamine) linked by
1
4-glycosidic linkages and covalently bound to serine residues of
the core protein. It has various O-sulfonate,
N-sulfonate, and N-acetyl substituents that are
usually heterogeneously distributed along the GAG chains.
Protein core.
The protein cores of HP are diverse and heterogeneous, vary in size
from 20 to 150 kDa, and appear to share only the capacity to bear GAG
chains. Repetitive serine-glycine sequences are found in the protein
core of heparin. The GAG chains are connected to the protein core
through a tetrasaccharide
(D-GlcA-D-Gal-D-Gal-D-Xyl) known as the linkage region (Fig. 2)
(26). HP helicity (i.e., secondary structural pattern or
coiling of the GAG chains) in conjunction with chirality (i.e.,
configuration of the carbon atoms at the asymmetric centers present in
HP) inherent in the constituent monosaccharides makes it a unique
macromolecule. Low molecular weight (LMW) HPs currently used in the
treatment of acute proximal deep vein thrombosis are obtained by
cleaving the GAG chains from the protein core.

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Fig. 2.
Structure of heparin (HP) demonstrating the peptide core with
glycosaminoglycan chains attached through a linkage region
tetrasaccharide: glucuronic acid (GlcA)-galactose (Gal)-Gal-xylose
(Xyl).
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Differences in the structure of HP and heparan sulfate.
HP and heparan sulfate (HS) originate from the same biosynthetic
precursor, N-acetylheparosan (Fig.
3). After the initial assembly of the
N-acetylheparosan polymer from monosaccharide precursors,
biosynthesis proceeds much further for HP than for HS. The following
steps are involved in the biosynthesis of heparin: 1)
N-deacetylation followed by N-sulfation of the basic sugar glucosamine
(GlcN), 2) glucuronate C-5 epimerization of the acidic sugar
D-glucuronic acid (GlcA) to L-iduronic acid
(IdoA), and 3) further O-sulfation of the acidic and basic
monosaccharides compared with those for HS (34, 37, 46, 47,
53).

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Fig. 3.
A: sequence of polymer modification steps involved in
the biosynthesis of HP. B: location and possible
substitution groups for each sugar moiety. Top: disaccharide
formula represents repeating disaccharide unit of
N-acetylheparosan.
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These additional biosynthetic steps for HP result in a GAG with a
content of sulfamino groups that exceeds its content of acetamido
groups and the concentration of the O-sulfate groups exceeds
that of N-sulfonate groups. Only such GAGs qualify to be
called HP. All other polysaccharides are known as HSs
(23). Twenty-four different disaccharide structures
(IdoA/GlcA-GlcN) are possible from the combination of different sulfate
residues in HP/HS GAG chains (Fig. 4).

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Fig. 4.
Different substitutions of sugar moieties (glucuronic acid,
iduronic acid, and glucosamine) in HP/heparan sulfate.
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HP AND PULMONARY HYPERTENSION |
Thompson and Hales (66) have recently
reviewed the effect of HP on pulmonary hypertension and the associated
vascular remodeling. Briefly, in our laboratory, Hales et al.
(29) have shown in a mouse model of chronic hypoxia that
HP inhibited the medial smooth muscle increase in vessels associated
with terminal bronchioles, reduced right ventricular systolic pressure,
and partially prevented the increase in medial thickness of
intra-acinar vessels after 26 days of hypoxia. HP did not alter the
hematocrit and was effective at low doses that did not prolong the
partial thromboplastin time. HP did not block the rise in right
ventricular systolic pressure after acute hypoxia, indicating that HP
prevented vascular remodeling through a mechanism that did not involve
blockade of hypoxic vasoconstriction.
Subsequently, in a guinea pig model of chronic hypoxia pulmonary
hypertension (67), Hassoun et al. (33) showed
that certain commercial HP preparations given by continuous
subcutaneous infusion resulted in a 50% reduction in medial thickness
of alveolar duct vessels (Fig. 5) and
completely prevented the medial smooth muscle increase in vessels
associated with terminal bronchioles (Fig. 6). Moreover, our laboratory found that
fully established hypoxic pulmonary hypertension in the guinea pig was
substantially reversed by HP (32) and that HP by aerosol
was effective (64). Our laboratory has also shown that HP
lots even from the same company vary in their ability to inhibit SMC
proliferation and hypertrophy (44) and that this variation
correlates with the ability of these HPs to prevent hypoxic pulmonary
hypertension (68). Rats were said to be resistant to HP,
but Du et al. (22) found that a strongly antiproliferative
HP was effective in rats.

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Fig. 5.
A: distal pulmonary artery with 2 discernible elastic
laminae and a thick media from a hypoxic control animal. The artery is
adjacent to a terminal bronchiole. B: distal pulmonary
artery with 2 elastic laminae from a HP-treated hypoxic animal. Here
the media is significantly thinner. Elastin stain; original
magnification, ×125. [Reprinted from Hassoun et al.
(33). Official Journal of the American Thoracic Society.
Copyright 1989, American Lung Association.]
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Fig. 6.
A: distal pulmonary artery accompanying an alveolar duct
from a hypoxic control animal. Two elastic laminae separated by a
thickened media are seen. B: distal pulmonary artery
accompanying an alveolar duct from a HP-treated hypoxic animal. There
is scant smooth muscle present between the 2 elastic laminae. Elastic
stain; original magnification, ×500. [Reprinted from Hassoun et al.
(33). Official Journal of the American Thoracic Society.
Copyright 1989, American Lung Association.]
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ANTIPROLIFERATIVE ACTIVITY OF HP AND ITS DERIVATIVES ON
PASMCS |
Mechanisms contributing to HP inhibition of SMC growth.
HP is a potent inhibitor of SMC proliferation (9, 28, 35,
68). Both anticoagulant and nonanticoagulant HP are reported to
possess effective SMC antiproliferative activity (55).
Although much attention has been focused on factors that stimulate SMC proliferation (62), very little is known about the
mechanisms maintaining these cells in a quiescent state or about the
reestablishment of a quiescent state after their proliferative response
has been initiated.
Circulating HP binds to endothelial cells and is taken up by the
reticuloendothelial system where it enters a cellular pool to be
released at a later stage (38). Furthermore, HP binds to
specific binding sites on SMCs and is internalized (14). Some antiproliferative effects are mediated by specific binding, although it is not clear whether internalization is essential. HP
blocks the cell cycle at either the
G0/G1 transition point (12)
or at mid to late G1 progression (14, 49, 58)
and may inhibit such cellular intermediate processes as protein kinase C activation, c-Fos and c-Myc induction (13, 77),
activator protein-1/Fos-Jun binding activity, and posttransitional
modification of Jun B (2, 7, 54). HP has also been shown
to selectively block the protein kinase C pathway of mitogenic
signaling (20) and the phosphorylation of
mitogen-activated protein kinase (52).
We have demonstrated that PASMC mitogens such as platelet-derived
growth factor and epidermal growth factor act through the Na+/H+ antiporter by stimulating a one-for-one
exchange of extracellular Na+ for intracellular
H+ to cause intracellular alkalinization, a permissive
first step for cell division (56). Furthermore, Dahlberg
et al. (19) have demonstrated that antiproliferative HPs
block Na+/H+ exchange in a manner directly
related to antiproliferative activity.
Structure-function relationship.
To understand the structure-function relationship of the HP
polysaccharide, we compared the antiproliferative activity of three
commercially available HPs. These preparations were from Upjohn,
Elkins-Sinn, and Choay Pharmaceuticals. The growth-inhibitory activity
of these HPs on PASMCs varied and was in the order Upjohn > Elkins-Sinn > Choay (19). The properties of
these HP preparations are summarized in Table
2.
3-O-sulfonation of glucosamine residue is not critical for
antiproliferative activity.
These three commercially available HPs were degraded with heparitinases
I and II to evaluate the overall content of 3-O-sulfation of
glucosamine in whole HPs to see whether the 3-O-sulfate
content correlated with the antiproliferative effect. These enzymes
were unable to degrade the components of HP containing
3-O-sulfate on the glucosamine residue into disaccharide
units, and instead, tetrasaccharides were formed (Fig.
7) (79). Thus the
tetrasaccharide content in the digest correlates with the content of
3-O-sulfate. The oligosaccharide profiles of the three
batches of HPs after digestion with heparitinases I and II demonstrated
that the most potent HP (Upjohn) contained the least amount of
tetrasaccharide, i.e., the least amount of 3-O-sulfate on
glucosamine residues. These results suggest that the presence of
3-O-sulfonated glucosamine residues in whole HP are not an essential
requirement for antiproliferative activity as previously reported
(11) based on data derived from the synthetic
pentasaccharide (63). The
-disaccharides [hexauronic acid (HUA)-glucosamine (GlcN)] liberated after heparitinase
treatment of these three HPs were analyzed by us, and the
results are given in Table 3
(25). The results show that the most potent Upjohn HP
preparation had the largest amount of trisulfonated
-disaccharide.

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Fig. 7.
A: HP sequences with different sulfonation
patterns arbitrarily assigned. Solid arrows, cleavage; broken arrow, no
cleavage by heparitinases I and II; *, O-substituent inhibits the
cleavage by enzyme treatment. B: cleavage pattern of
heparin containing 3-O-sulfonate glucosamine residues by
heparitinases I and II. [Reprinted from Garg et al. (25). Copyright
1996, Academic Press.]
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Influence of molecular weight protein core and GAGs of HP on
antiproliferative activity.
Structure-function studies carried out by preparing discrete sizes of
antiproliferative HP fragments by chemical modification of HP show that
dodecasaccharide and larger fragments had maximal antiproliferative
activity (10, 72). Furthermore, Tiozzo et al.
(72) demonstrated that the reduction in the molecular
weight (MW) of HP is associated with a progressive reduction in the
antiproliferative activity. These studies were based on chemically
modified HP. In recent years, Joseph et al. (39) attempted
to assess the influence of MW, protein core, and GAG chain of native HP
on PASMC proliferation. The most potent Upjohn batch of HP was
fractionated by dissolving it in water and dialyzing it against water
without chemical depolymerization. This was followed by lyophilization of both the dialyzate (giving a LMW HP fraction of <3.5 kDa) and the
HP fraction retained in the dialysis bag [yielding a high molecular
weight (HMW) HP fraction of >3.5 kDa]. The core protein of Upjohn HP
was isolated by treatment with heparitinases I and II
(25). GAG chains of Upjohn HP were liberated with alkaline borohydride treatment (8). No appreciable difference on
the growth inhibition of PASMCs between the LMW and HMW HP fractions was found. The protein core showed no antiproliferative activity. The
GAG chain had similar inhibition on the growth inhibition of PASMCs as
that of the parent HP. These data suggest that the antiproliferative
properties of HP reside in the GAG chain and not in the core protein.
These data also suggest that both the HMW and LMW HP fractions have
sufficient N- and O-substituents in the carbohydrate residues necessary
for the antiproliferative effect.
Effect of sulfonation of polysaccharides on antiproliferative
activity.
The effect of N- and O-linked sulfate groups on glucosamine and
uronic sugar residues in HP on the antiproliferative activity has been
studied in several laboratories. To this purpose, Tiozzo et al.
(71) modified HP to produce N-desulfated and O-desulfated HP derivatives. They found that 2-O-sulfonation in HP is important for
antiproliferative properties (37). On the other hand,
Wright et al. (75) reported that 2-O-sulfonation in HP is
not essential for antiproliferative activity. To clarify the role of N-
and O-sulfation in PASMC growth inhibition, we fully sulfonated HP, HS,
chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA)
using sulfur trioxide (Fig. 8,
A-E) (45, 51). N-sulfonated acharan sulfate was prepared by N-deacetylation and N-sulfonation of
acharan sulfate (Fig. 8F) (41, 78). All these
derivatives were analyzed for PASMC antiproliferative activity (Fig.
9) (24).

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Fig. 8.
Major and variable sequences of original and fully
sulfonated glycosaminoglycans. A: HP. B: heparan
sulfate. C: chondroitin sulfate. D: dermatan
sulfate. E: hyaluronan. F: acharan sulfate and
N-sulfoacharan sulfate. X, H or SO3; Y, acetyl or
SO3. [Reprinted from Garg et al. (25).
Copyright 1996, Academic Press.]
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Fig. 9.
Effect of various polysaccharides on bovine pulmonary
artery smooth muscle cells (BPASMC) grown in medium containing 10%
fetal calf serum and either native (original) or fully sulfonated
glycosaminoglycan, acharan sulfate (far right solid
bar), or N-sulfoacharan sulfate (far
right open bar). Samples were standardized to cell growth in
medium containing 10% serum without polysaccharide.
+Significant reduction in cell growth compared with the
standard, P < 0.05. ++Significant increase
in cell growth compared with the standard, P < 0.05. *Significant reduction in cell growth compared with standard,
P > 0.05. **Significant reduction in cell growth
compared with both the standard and the native polysaccharide,
P > 0.05. #Significant reduction in cell
growth compared with native acharan sulfate (P > 0.05)
but not with the standard. [Reprinted from Garg et al.
(25). Copyright 1996, Academic Press.]
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HP and HS.
Fully sulfonated HP did not produce any greater growth inhibition of
PASMCs than did the parent HP, indicating that native HP already has
the necessary number of N- and O-sulfonate groups to produce maximum antiproliferative activity (Fig. 9) However, fully
sulfonated HS containing both N-acetyl and
N-sulfonate substituents suppressed the growth of PASMCs to
a greater degree than the parent HS, showing that HS does not have
sufficient O-sulfonate groups for full antiproliferative potency.
HA.
The antiproliferative activity of native HA (Fig. 9) became strikingly
significant after sulfonation and equaled that of native HP. Because
HA, like CS and DS, has a 1
3 linkage between sugars, whereas HP and
HS have a 1
4 linkage, the linkages between acidic and basic sugars
(Fig. 8, A and E) do not seem to be critical for
antiproliferative activity.
Acharan sulfate.
Neither acharan sulfate nor N-sulfoacharan sulfate had any
antiproliferative activity, showing that sulfation alone does not produce antiproliferative activity.
CS and DS.
Full sulfonation of CS and DS reversed their proliferative effect on
PASMCs and produced an antiproliferative effect similar to HP (Fig. 9).
Because both CS and DS have a variable sequence that is not sulfonated,
this shows that O-sulfonation of both types of sugar residues of the
GAG is necessary for the antiproliferative activity. Because both CS
and DS GAGs consist of N-acetylgalactosamine residues only,
the above data also suggest that N-sulfonated glucosamine residues in
HP are replaceable. Furthermore, because all the basic sugar residues
are N-acetylated in CS and DS, this suggests that the N-sulfonated
basic sugar residues are not critical for antiproliferative activity.
Anticoagulant activity.
The anticoagulant activity of fully sulfonated HP was significantly
reduced compared with the anticoagulant activity of native HP. All the
other sulfonated GAGs showed very little anticoagulant activity
(24). This demonstrates that the structural determinants of HP for its anticoagulant and antiproliferative activities are unrelated.
In summary, the above studies on the effects of HP and its
derivatives on PASMC antiproliferative properties show that
1) 3-O-sulfonate substitution of glucosamine
residues is not critical in whole HP for antiproliferative activity,
2) the HMW and LMW of a given HP do not affect the potency,
3) the antiproliferative properties of HP reside in the GAG
chain and not in the core protein, 4) a certain number of
O-sulfonate groups of HP is essential for the full
antiproliferative effect of HP, 5) the
N-sulfonate group on basic sugar residues is not critical
for antiproliferative activity, 6) the basic sugar residues
of glucosamine are replaceable with galactosamine residues,
7) the anomeric linkage of acidic and basic sugar residues
is not critical for antiproliferative activity, 8) the
commercially available HPs have a varying degree of antiproliferative
activity on PASMCs, and 9) the antiproliferative and
anticoagulant activities reside in different domains of HP.
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CONCLUDING REMARKS |
The biosynthesis of HP chains is initiated by the formation
of a (GlcA
1
4GlcNAc
1
4)n polymer (Fig.
3) that is subsequently modified. Various modification reactions are
generally incomplete in the sense that only a fraction of the potential
substrate residues is utilized at each step. These processes therefore
lead to sequence heterogeneity of HP. Functional properties of HP and
other proteoglycans depend heavily on their ability to bind receptors.
HP binds with receptors in a selective manner. By virtue of this
property, HP possesses different types of biological activities. An
increase in antiproliferative activity in fully sulfonated HS, DS, CS, and HA shows a potential for the development of one of these
derivatives as a therapeutic agent for the treatment of vascular
remodeling in the near future.
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ACKNOWLEDGEMENTS |
Work in our laboratory is supported by National Heart, Lung, and
Blood Institute Grant HL-39501.
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FOOTNOTES |
Address for reprint requests and other correspondence: C. A. Hales, Pulmonary/Critical Care Unit, Harvard Medical School, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-1111 (E-mail: chales{at}partners.org).
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.
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