1. No category

CHEST
Original Research
PULMONARY HYPERTENSION
Low-Molecular-Weight Heparin Inhibits
Hypoxic Pulmonary Hypertension and
Vascular Remodeling in Guinea Pigs*
Essam Al-Ansari, MD, MPH; Hong-kai Du, MD; Lunyin Yu, MD;
Cristhiaan D. Ochoa, MD; Hari G. Garg, PhD; Deborah A. Quinn, MD;
and Charles A. Hales, MD
Rationale: We have shown previously that antiproliferative unfractionated heparins block
hypoxia-induced pulmonary arterial hypertension (PAH) and vascular remodeling, and hypothesized that low-molecular-weight heparins (LMWHs) would too.
Objectives: To determine the potential role and mechanisms of dalteparin and enoxaparin (two
LMWHs) in inhibiting hypoxic PAH and vascular remodeling.
Methods: Male Hartley guinea pigs were exposed for 10 days to normobaric 10% oxygen with
dalteparin (5 mg/kg), enoxaparin (5 mg/kg), or with an equivalent volume of normal saline
solution. Normoxic control animals (n ⴝ 5) received room air for 10 days. Bovine pulmonary
artery smooth-muscle cells (PASMCs) were grown in 10% fetal bovine serum without heparin,
with dalteparin (1 ␮g/mL) or with enoxaparin (1 ␮g/mL).
Measurements: Pulmonary arterial pressure (PAP), cardiac index, right ventricular heart weight
divided by left ventricular plus septum weight (RV/LVⴙS), hematocrit, percentage of wall thickness
of intraacinar vessels (%WT-IA), percentage of wall thickness of terminal bronchiole vessels
(%WT-TA), and the percentage of thick-walled vessels (%Thick) were determined. In PASMCs,
expression of p27 and cell growth were compared because in mice whole heparin depends on p27 for
its antiproliferative action.
Main results: In hypoxic animals, hematocrit, PAP, total pulmonary vascular resistance index,
RV/LVⴙS, %WT-IA, %WT-TA, and %Thick all rose significantly vs normoxic control animals
(p < 0.05); cardiac index was unchanged. Dalteparin but not enoxaparin significantly reduced PAP,
total pulmonary vascular resistance index, and RV/LV ⴙ S (p < 0.05 vs hypoxia alone); inhibited
PASMC growth; and upregulated p27 expression. Enoxaparin moderately reduced vascular remodeling, which did not translate into less pulmonary hypertension.
Conclusions: Not all LMWHs are the same. Dalteparin was more effective than enoxaparin in
inhibiting pulmonary hypertension and vascular remodeling in hypoxic guinea pigs.
(CHEST 2007; 132:1898 –1905)
Key words: dalteparin; enoxaparin; hypoxia; low-molecular-weight heparin
Abbreviations: FBS ⫽ fetal bovine serum; LMWH ⫽ low-molecular-weight heparin; PAH ⫽ pulmonary arterial hypertension; PAP ⫽ pulmonary arterial pressure; PASMC ⫽ bovine pulmonary artery smooth-muscle cell; RV ⫽ right ventricular;
RV/LV ⫹ S ⫽ right ventricular heart weight divided by left ventricular plus septum weight; SMC ⫽ smooth-muscle cell;
TPVRI ⫽ total pulmonary vascular resistance index; %Thick ⫽ percentage of thick-walled vessels; %WT-IA ⫽ percentage of
wall thickness of intraacinar vessels; %WT-TA ⫽ wall thickness of the terminal bronchiolar arterioles
espite recent advances, pulmonary arterial hyperD tension
(PAH) remains a major cause of morbidity
and mortality.1 Chronic hypoxia in addition to causing
vasoconstriction leads to the development of pulmonary vascular remodeling with changes that include
hyperplasia, hypertrophy, and distal accumulation of
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pulmonary artery smooth-muscle cells (PASMCs) in
the vascular bed.2– 4 These changes develop in guinea
pigs exposed to chronic hypoxia.5–7
Heparin, a linear acidic polysaccharide that was
discovered nearly 90 years ago, has a potent inhibitory effect on PASMC growth in vitro and in vivo.8
Original Research
We and others7–10 have found that different commercial heparin preparations vary in their effectiveness. We have shown that select batches of Upjohn
heparin (Pharmacia Upjohn; Kalamazoo, MI) were
effective in inhibiting PASMC growth, PAH, and
vascular remodeling.9 The inhibition of pulmonary
vascular remodeling and PASMC growth was dependent on heparin-induced upregulation of p27, a cell
cycle-progression inhibitor.11
The discovery and introduction of low-molecularweight heparins (LMWHs) have enhanced the safety
of heparin therapy.12 They are easier to administer
than unfractionated heparin. LMWH was found in a
porcine acute lung injury model to have protective
effects due to its effects on neutrophil adhesion,
tumor necrosis factor-␣ receptor stabilization, and
the decrease of thromboxane B2 production.13 It is
well established that different LMWHs vary in their
physical and chemical properties due to differences
in their methods of production (Table 1).14 These
differences can translate into differences in pharmacodynamic and pharmacokinetic properties.15
Enoxaparin and dalteparin are approved in the
United States for clinical use. We hypothesized that
subcutaneous injections of either enoxaparin, dalteparin, or both would inhibit PAH and vascular remodeling in guinea pigs exposed to chronic hypoxia, and the
inhibition of pulmonary vascular remodeling would
depend on the degree of p27 expression. Therefore, we
injected guinea pigs exposed to 10 days of 10% oxygen
with either enoxaparin or dalteparin subcutaneously,
and assessed pulmonary hemodynamics and vascular
remodeling at the end of hypoxia, compared to normoxic or hypoxic controls injected with normal saline
solution.
Materials and Methods
Isolation and Culture of Bovine PASMC
Bovine main pulmonary arteries were obtained from a local
slaughterhouse. Isolation and culture of PASMCs were performed as previously described.9 Cells were harvested and stored
*From the Pulmonary and Critical Care Unit, Department of
Medicine, Massachusetts General Hospital, Harvard Medical
School, Boston, MA.
This work was supported by National Institutes of Health grants
HL39150 (Dr. Hales) and HL03920 (Dr. Quinn), and American
Heart Association grant 0525926T (Dr. Al-Ansari).
The authors have no conflicts of interest to disclose.
Manuscript received April 7, 2006; revision accepted August 1,
2007.
Reproduction of this article is prohibited without written permission
from the American College of Chest Physicians (www.chestjournal.
org/misc/reprints.shtml).
Correspondence to: Charles A. Hales, MD, Massachusetts General Hospital, Pulmonary and Critical Care Unit, 55 Fruit St,
Bullfinch 148, Boston, MA 02114; e-mail: [email protected]
DOI: 10.1378/chest.06-0941
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Table 1—LMWH and the Method of Production
Trade
Name
Approved
Name
Fluxum
Parnaparin
sodium
Fragmin
Dalteparin
sodium
Enoxaparin
sodium
Certiparin
sodium
Ardeparin
sodium
Lovenox
Sandoparin
Normiflo
Manufacturer
Opocrin s.p.a,
alfa
wasserman
Pharmacia
Upjohn
Aventis
Sandoz AG
Wyeth-Ayerst
Research,
Pharmacia,
Hepar
Method of
Production
Peroxidolysis
Deaminative
cleavage
Chemical ␤
elimination
Deaminative
cleavage
Peroxidolysis
in liquid nitrogen. Cells from passages three to six were used in
this experiment. Identification of the cells as smooth-muscle cells
(SMCs) was done by demonstrating typical morphology and by
the presence of a ␣ smooth-muscle actin.
PASMC Growth Assay
To assay for the antiproliferative activity of heparin, 1.5 ⫻ 105
PASMCs were placed onto 60-mm tissue culture dishes containing 3 mL of our standard medium: RPMI-1640 (Media Tech;
Washington, DC) with 100 ␮g/mL penicillin, 100 ␮g/mL streptomycin, and 10% fetal bovine serum (FBS) [Hazelton Biologics;
Lenexa, KS]. After 48 h, the cells were growth arrested by
decreasing the serum concentration of the culture medium to
0.1% FBS. Following 48 h of the growth-arrested phase, the cells
were classified into treatment groups as follows: standard medium ⫹ 0.1% FBS, standard medium ⫹ 10% FBS, and standard
medium containing either dalteparin, or enoxaparin ⫹ 10% FBS
(five wells per treatment). Heparins were tested at concentrations of 1 ␮g/mL. Experiments were repeated three times for
each heparin. Cell viability was determined by trypan blue
exclusion. Percentage of growth was calculated as follows: (net
cell growth in treated medium/net cell growth in standard
medium) ⫻ 100, where net cell growth ⫽ cell growth in standard
or treated medium ⫺ cell growth in growth arrest medium, as
previously described.9
Experimental Animals and Hypoxic Chambers
Male Hartley guinea pigs weighing from 300 to 450 g were
obtained from the Charles River Laboratories (Wilmington, MA).
We produced normobaric hypoxia by venting room air (6 L/min)
into a 219-L polymethyl methacrylate box and mixing it with
nitrogen (5 L/min). This produced an oxygen level of 10%.
Carbon dioxide absorbent was added to keep the carbon dioxide
content ⬍ 0.5%. Gas was circulated in the chamber with a fan,
and gas samples were tested daily.
Catheter Placement, Hemodynamic Measurements, and Blood
Gas Analysis
Guinea pigs were anesthetized by intraperitoneal administration of ketamine (40 mg/kg) and diazepam (0.01 mg/kg). Rightsided heart pressure measurements were obtained on room air
1 h after removal of the animal from hypoxia by introducing a
silicone tube (0.012-inch inner diameter, 0.021-inch outer diamCHEST / 132 / 6 / DECEMBER, 2007
1899
Histologic Grading of Pulmonary Hypertension
The animals were killed by an overdose of interperitoneal ketamine and diazepam. A simultaneous fixing of the lungs was done
using 10% buffered formalin. We assessed in a blinded fashion the
extent of pulmonary vascular remodeling by measuring the percentage of thick-walled vessels (%Thick) and the percentage of wall
thickness as previously reported.16 The right ventricular (RV) free
wall was removed using a modified procedure of Fulton et al.17 We
measured the diameter of the vessels (distance between external
elastic lamina) and the medial wall thickness (distance between the
internal elastic lamina and the external elastic lamina) using an
ocular micrometer. Vessels were considered thick walled if they
contained an internal and external lamina for ⬎ 50% of the circumference of the vessel. %Thick was expressed as the number of
thick-walled intraacinar vessels divided by the number of thickwalled plus thin-walled vessels ⫻ 100. Percentage of wall thickness
was defined as the medial wall thickness divided by the diameter of
the vessel ⫻ 100. We assessed RV hypertrophy by measuring the
ratio of the dry RV free wall weight to the dry weight (RV heart
weight divided by left ventricular plus septum weight [RV/LV ⫹ S]).
Experimental Design
We had two main study groups: hypoxic and normoxic animals.
The hypoxic group was put in 10% hypoxia for 10 days, while the
normoxic animals breathed room air for the same duration. The
hypoxic group was divided into three subgroups: (1) hypoxic plus
dalteparin (received daily subcutaneous injection of dalteparin, 5
mg/kg, or 2 mg/kg for 10 days, lot number 94082A62), (2) hypoxia
plus enoxaparin (received daily subcutaneous injection of enoxaparin, 5 mg/kg, for 10 days, lot No. 89339), and (3) hypoxic and
normoxic control animals received daily subcutaneous injections
of an equivalent volume of normal saline solution for 10 days. An
additional hypoxia-treatment group was added in this current
work to test the dose response effect for dalteparin (received
daily subcutaneous injection of dalteparin, 2 mg/kg, for 10 days).
No bleeding was observed in either the enoxaparin or dalteparin
treatment groups, and partial thromboplastin time was not
different between the two groups, (27 ⫾ 1 s vs 35 ⫾ 2 s, respectively; p ⬎ 0.05) nor from control animals (29.7 ⫾ 0.4 s). At the
end of 10 days, catheters were placed under anesthesia for
measurement of pulmonary hemodynamics while the animals
were spontaneously breathing room air.
Statistical Evaluation
Statistical analysis were performed using statistical software
(Statview 5.0; Abacus Concepts; Berkeley, CA). Significance level
was set at p ⬍ 0.05, and all values were expressed as mean ⫾ SE.
We compared the pulmonary hemodynamic measurements, percentage of wall thickness, and %Thick by using analysis of
variance. If analysis of variance findings were significant, multiple
comparisons were made among groups using the Fisher protected least-significant difference test.
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Results
Pulmonary Hemodynamics and Arterial Blood Gas
Measurements
After 10 days of 10% oxygen, pulmonary artery
pressure (PAP) and total pulmonary vascular resistance index (TPVRI) [mean PAP/cardiac index] rose
significantly (p ⬍ 0.05 vs normoxic control animals)
in all animal groups, while cardiac index was unchanged (p ⬎ 0.05 vs normoxic control animals) [Fig
1, Table 2]. Dalteparin (5 mg/kg) reduced PAP and
TPVRI significantly, whereas enoxaparin (5 mg/kg)
was without significant effect (Fig 1). Cardiac index
did not change significantly among these groups
(p ⬍ 0.05) [Table 2]. In the hypoxic animals treated
with a lower dose of dalteparin (2 mg/kg; n ⫽ 5),
PAP (26 ⫾ 5; p ⫽ 0.34) and TPVRI (0.075 ⫾ 0.01;
p ⫽ 0.8) were not significantly reduced when compared to hypoxic control animals. Cardiac index did
not change significantly in this group when compared to hypoxic control animals or other groups of
LMWH treatment. Blood gases drawn showed lower
Pao2 vs normoxic control animals (p ⬍ 0.05) [Table
3]. There was no evidence of breathing difficulty in
the hypoxic groups of animals vs the normoxic
animals. There was no significant difference in Pao2,
Paco2, or pH among guinea pigs in the hypoxic
control, dalteparin-treated, or enoxaparin-treated
animals.9
RV Hypertrophy and Hematocrit
Animals exposed to 10 days of hypoxia had significant RV hypertrophy as measured by RV/LV ⫹ S
dry weight (Fig 2). RV/LV ⫹ S ratio was significantly
reduced in animals treated with dalteparin (5 mg/kg)
but not enoxaparin, as compared with hypoxic con-
* p < 0.05 vs. normoxic control
# p < 0.05 vs. hypoxic control
P u lm o n a ry A rte ria l P re s s u re
( mm Hg)
eter) via a right neck cutdown to the right external jugular vein.5,9
The catheter was connected to a transducer (Model 049924-507;
Cobe Laboratories; Lakewood, CO) that was connected to an
amplifier (Gould monitor, model RS3200; Gould; Cleveland,
OH). We confirmed the position of the catheter by the pulse
tracing on an oscilloscope. In order to measure the cardiac output
by thermodilution, we placed a 1.5F thermal dilution probe
(model EX 121003; American Edwards Laboratories; Irving, CA)
into the thoracic aorta through a right internal carotid artery
cutdown. Blood gases were drawn on anesthetized guinea pigs
1 h after removal from the chronic hypoxia chamber.
35
30
*
*
*
#
25
20
15
10
5
0
Figure 1. PAP in guinea pigs after 10 days of hypoxia treated
with either enoxaparin or dalteparin. Animals exposed to 10%
hypoxia and which received normal saline solution injections
served as hypoxic control animals. Normoxic control animals
breathed room air for 10 days. The number of animals in each
group is given in Table 2.
Original Research
Table 2—PAP, Cardiac Index, and TPVRI in Study
Groups*
Groups
Normoxic control
(n ⫽ 5)
Hypoxic control
(n ⫽ 8)
Hypoxia plus
enoxaparin (n ⫽ 7)
Hypoxia plus
dalteparin (n ⫽ 8)
* p < 0.05 vs. normoxic control
# p < 0.05 vs. hypoxic control
10 ⫾ 1
337 ⫾ 10
0.030 ⫾ 0.002
28 ⫾ 2†
365 ⫾ 25
0.078 ⫾ 0.009†
28 ⫾ 1†
335 ⫾ 10
0.073 ⫾ 0.006†
22 ⫾ 1†‡
386 ⫾ 34
0.058 ⫾ 0.003†
*
*
PAP, Cardiac Index,
TPVRI,
mm Hg
mL/min/kg mm Hg/mL/min/kg
* #
Figure 2. RV hypertrophy measured as RV/LV ⫹ S in guinea
pigs after 10 days of 10% hypoxia treated with either enoxaparin
or dalteparin. Animals exposed to 10% hypoxia and which
received normal saline solution injections served as hypoxic
control animals. Normoxic control animals breathed room air for
10 days. *p ⬍ 0.05 vs normoxic control; #p ⬍ 0.05 vs hypoxic
control. The number of animals in each group is given in Table 2.
*Data are presented as mean ⫾ SE.
†p ⬍ 0.05 vs normoxic control.
‡p ⬍ 0.05 vs hypoxic control.
trol (p ⬍ 0.05), but it was still higher than in normoxic control animals (p ⬍ 0.05) [Fig 2]. Hematocrit
was significantly increased after hypoxia, and was
unchanged with treatment with either enoxaparin or
dalteparin (Fig 3).
Pulmonary Vascular Remodeling
In animals exposed to 10 days of hypoxia, there was
significant pulmonary vascular remodeling, which was
measured as percentage of wall thickness of intraacinar
vessels (%WT-IA), percentage of wall thickness of
terminal bronchiolar arterioles (%WT-TA), and
%Thick intraacinar vessels (Table 4). Pulmonary vascular remodeling, as shown by %WT-IA, %WT-TA,
and%Thick, was significantly less in the animals treated
with enoxaparin and dalteparin than in the hypoxic
control animals (Table 4, Fig 4, top left, A, through
top right, D). The remodeling in the dalteparin- and
enoxaparin-treated animals was not statistically different for both %WT-IA and %Thick measurements,
although there was a trend for less remodeling in the
dalteparin animals. There was a statistically significant
difference in the %WT-TA between dalteparin- and
enoxaparin-treated animals (p ⬍ 0.05).
LMWH Inhibition of PASMC Growth
Dalteparin inhibited PASMC growth (n ⫽ 5)
and upregulated p27 expression (n ⫽ 3), whereas
enoxaparin stimulated PASMC growth (n ⫽ 5) and
had no significant effect on p27 expression (n ⫽ 3)
[Fig 5].
Discussion
Chronic hypoxia leads to PAH through vasoconstriction and vascular architectural changes, and an increase
in hematocrit.18,19 Heparin, an endogenous glycosaminoglycan, has been shown to inhibit PASMC proliferation.9,20 –23 Therefore, heparin has the potential to
influence pulmonary vascular remodeling through its
Table 3—Arterial Blood Gas Determinations in Guinea
Pigs After 10 Days of Hypoxia in the Chamber With
Either Enoxaparin or Dalteparin Treatment*
Groups
Pao2,
mm Hg
Paco2,
mm Hg
pH
Normoxic control
Hypoxic control
Hypoxia plus enoxaparin
Hypoxia plus dalteparin
91 ⫾ 3
83 ⫾ 2
83 ⫾ 2†
82 ⫾ 2†
37 ⫾ 2
35 ⫾ 1†
36 ⫾ 1†
35 ⫾ 1†
7.42 ⫾ 0.02
7.40 ⫾ 0.01
7.40 ⫾ 0.05
7.40 ⫾ 0.02
*Data are presented as mean ⫾ SE. Animals exposed to hypoxia and
receiving normal saline solution injections served as hypoxic control
animals. Normoxic control animals breathed room air for 10
days (n ⫽ 5 for all groups).
†p ⬍ 0.05 vs normoxic control.
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H em atocrit
(% )
* p < 0.05 vs. normoxic control
80
70
60
50
40
30
20
10
0
*
*
*
Figure 3. Hematocrit in guinea pigs after (n ⫽ 5 for each group)
10 days of 10% hypoxia treated with either enoxaparin or
dalteparin. Animals exposed to 10% hypoxia and which received
normal saline solution injections served as hypoxic control animals. Normoxic control animals breathed room air for 10 days.
*p ⬍ 0.05 vs normoxic control.
CHEST / 132 / 6 / DECEMBER, 2007
1901
Table 4 —Measurement of Wall Thickness and Percentage of Thick-Walled Pulmonary Vessels*
Groups
Normoxic
Control (n ⫽ 5)
Hypoxic
Control (n ⫽ 5)
Hypoxia Plus
Enoxaparin (n ⫽ 5)
Hypoxia Plus
Dalteparin (n ⫽ 5)
%WT-TA
%WT-IA
%Thick†
14.2 ⫾ 1.2
10.6 ⫾ 0.7
11.9 ⫾ 1.2
38.4 ⫾ 2.3†
19.9 ⫾ 1.7‡
61.1 ⫾ 3.8‡
29.4 ⫾ 1.9†§
15 ⫾ 0.9‡§
40 ⫾ 4‡§
22.7 ⫾ 2.3‡§储
12.2 ⫾ 1§
30.4 ⫾ 3.6‡§
*Data are presented as mean ⫾ SE.
†No. of thick-walled intraacinar vessels divided by the number of thick plus thin-walled vessels ⫻ 100.
‡p ⬍ 0.05 vs normoxic control.
§p ⬍ 0.05 vs hypoxic control.
储p ⬍ 0.05, dalteparin vs enoxaparin treatment.
effect on SMC proliferation.24 Heparin binds to specific binding sites on SMCs and is internalized.25
Heparin blocks the cell cycle at either G0/G1 transition
point,25 or at mid to late G1 progression.26 We have
shown in a mouse model of chronic hypoxia that
heparin inhibited the medial smooth muscle increase in
vessels associated with terminal bronchioles, reduced
the RV systolic pressure, and partially prevented the
increase in medial thickness of intraacinar vessels after
26 days of hypoxia.27 Similar results have been obtained
in rats and guinea pigs.7,9
Several studies8,9,20 have found that different commercial heparin preparations vary in their effect on
PASMC growth. We have shown that select batches
of Upjohn heparin were very effective in inhibiting
PASMC proliferation, PAH, and vascular remod-
eling.7,9,20,28,29 Since LMWHs need minimal or no
monitoring of their levels, have decreased risk of
heparin-induced thrombocytopenia,30 and decreased
risk of osteoporosis,31 we decided in this study to
explore the role of LMWHs in the inhibiting hypoxic
PAH compared to unfractionated heparin.
Compared with unfractionated heparin, LMWHs
have lower binding properties to plasma proteins,
which explain their increased half-life.32,33 They have
a bioavailability approaching 100% at low doses by
subcutaneous injections. They are polysulphated glycosaminoglycans with a mean molecular weight of 4
to 5 kd (range, 2 to 9 kd) and chain lengths of 12 to
18 sacharide units.11
The main objective of our present study was to
develop a potential therapeutic agent to prevent and
Figure 4. Representative photomicrographs of terminal bronchial (TA) and intraacinar (IA) vessels on
elastin stains of 5-␮m-thick sections of paraffin-embedded tissue. Top left, A, and bottom left, E:
Normoxic control animals. Top center left, B, and bottom center left, F: Hypoxic control animals
exposed to 10% hypoxia. Top center right, C, and bottom center right, G: Hypoxia plus enoxaparin
animals after 10 days of 10% hypoxia. Top right, D, and bottom right, H: Hypoxia plus dalteparin
animals after 10 days of 10% hypoxia (n ⫽ 5 in all four groups). Original magnification done at ⫻ 200
terminal bronchiole vessels, and at ⫻ 400 for intraacinar vessels. The number of animals in each group
is given in Table 2.
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Original Research
Figure 5. Bovine PASMCs were grown in 10% FBS media
without heparin (control), with dalteparin (1 ␮/mL), or with
enoxaparin (1 ␮/mL) for 72 h. At the end of exposure, percentage
of growth was determined (top, A), and cells were harvested for
western blot analysis of p27 expression (bottom, B). *p ⬍ 0.05 vs
control. GADPH ⫽ glyceraldehyde-6-phosphate dehydrogenase.
treat pulmonary vascular remodeling that occurs in
PAH. Enoxaparin and dalteparin were used at the
same dose level we had previously shown to be
effective when antiproliferative unfractionated heparin (Upjohn heparin), was used.9 We found in this
study that dalteparin and enoxaparin vary in their
ability to inhibit the growth of PASMC and pulmonary vascular remodeling, and that dalteparin was
more effective compared to enoxaparin. There was
no significant difference between the anticoagulation
profiles of both dalteparin and enoxaparin, and
hence the observed effect on pulmonary vasculature
could not be attributed to the anticoagulation effects.
Dalteparin, but not enoxaparin, was effective in
inhibiting PAH in these animals. PAP was reduced to
22 mm Hg, as compared with 28 mm Hg in hypoxic
control animals (p ⬍ 0.05) [Fig 1]. These animals
had a hematocrit of 69%. We as well as other
investigators34 have shown that if the hematocrit
were normal, the PAP would have been even lower.
We confirmed the antiproliferative effects of dalteparin in vitro and found that the antiproliferative
effects correlated with upregulation of p27, a cell
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cycle inhibitor, which we have found in mice to be
essential for antiproliferative effects of unfractionated heparin on SMCs.11
In this report, we used guinea pigs for the in vivo
test model and bovine PASMCs in vitro because
they are readily available and guinea pigs cells are
not. This difference in species may affect the results
and perhaps explain why in vivo in guinea pigs that
enoxaparin would moderately reduce pulmonary vascular remodeling but not significantly affect PASMC
growth or p27 production in vitro. More likely,
however, it is that the LMWH doses in vitro are only
an approximation of what is seen in the interstitial
fluid of in vivo PASMCs. We did not push up the
enoxaparin dose in vivo because it was already at a
level in excess of that clinically used, but if we had as
we have with unfractionated heparin,20 we may have
found an antiproliferative effect, just less potent than
that of dalteparin. We have also shown that heparin
inhibits pulmonary vascular remodeling through inhibition of the Na/H⫹ exchanger,20 and perhaps that
is important besides p27, although our mouse data
on p27 do not support that hypothesis.
Heparin may inhibit pulmonary vascular remodeling
by affecting other cell types in the lung, including
endothelial cells35 and fibroblasts.36 A previous in vitro
study by Khorana et al35 showed that both dalteparin
(average molecular weight, 5 kd) and enoxaparin (molecular weight, 4.2 kd) were tested in the inhibition of
endothelial cell tube formation in a gelatinous protein
mixture secreted by mouse tumor cells (Matrigel; BD
Biosciences; Franklin Lakes, NJ) in the presence of
fibroblast growth factor-2 stimulation. Dalteparin resulted in more effective inhibition of endothelial tube
formation (68% ⫾ 13%) as compared to enoxaparin
(46% ⫾ 3%) [p ⬍ 0.05]. Of interest is that the inhibition of heparin of endothelial cell proliferation was
dependent on the molecular weight of the heparin
products.35 Maximum inhibition of cell proliferation
occurred at approximately 6-kd molecular weight, with
less inhibition observed by both higher and lower
molecular-weight fractions. This suggests that the size
of the heparin may contribute to the antiproliferative
properties.
Additionally, enoxaparin is obtained by alkaline degradation of heparin benzyl ester from porcine intestinal
mucosa, whereas dalteparin is obtained by nitrous acid
depolymerization.14,28 The production of enoxaparin
maintains the internal structure of the parent heparin
glycosaminoglycan chains, with the exception of the
unsaturated nonreducing end. In contrast, the production of dalteparin removes part of their nonsulfated
uronic acid residues and, unlike enoxaparin and unfractionated heparin, also contains regions that remain
resistant to heparitinase II, suggesting other structural
modifications.37 During the preparation process of
CHEST / 132 / 6 / DECEMBER, 2007
1903
both dalteparin and enoxaparin, the protein core of
unfractionated heparin has been cleaved. We have
shown previously that the antiproliferative activity of
heparins resides in the glycosaminoglycan chains, and
not in the protein cores.38 Considerable amounts of
heparan sulfate glycosaminoglycan chains have been
shown to be present in dalteparin by using cellulose
plate electrophoresis, and it is likely that dalteparin and
enoxaparin differ in the amount of glycosaminoglycan
chains they contain. Heparan sulfate has a stronger
antiproliferative activity when compared to heparin.39
These structural variations result in different pharmacokinetic properties.
Furthermore, earlier studies37,38,40,41 of unfractionated heparin preparations have shown that partial desulfation and anionic charge pattern of these
compounds correlate with their antiproliferative
properties. Dalteparin in comparison to enoxaparin
has a less anionic charge group at the reducing end
of the molecule, thereby minimizing cellular interaction and protecting the compound from elimination.15 Fletcher et al42 have shown that both dalteparin and enoxaparin were effective in inhibiting
intimal hyperplasia developing in a prosthetic vascular patch graft implanted into sheep carotid arteries,
with dalteparin being more effective than enoxaparin. Clinical trials43,44 of LMWHs have shown differences in their hemorrhagic profiles, and therefore
they are not interchangeable in their anticoagulation
potency dose. In conclusion, our results suggest that
not all LMWHs have the same antiproliferative
effectiveness, and that dalteparin has a potential for
development as an effective drug for treatment for
hypoxic pulmonary hypertension, and possibly other
types of secondary pulmonary hypertension.
ACKNOWLEDGMENT: We gratefully acknowledge the work
of John Beagle for his assistance in the pulmonary artery
catheterization of the guinea pigs.
References
1 Farber HW, Loscalzo J. Pulmonary arterial hypertension.
N Engl J Med 2004; 351:1655–1665
2 Kourembanas S, Bernfield M. Hypoxia and endothelialsmooth muscle cell interactions in the lung. Am J Respir Cell
Mol Biol 1994; 11:373–374
3 Peacock A. Pulmonary hypertension due to chronic hypoxia.
BMJ 1990; 300:763
4 Tozzi CA, Poiani GJ, Harangozo AM, et al. Pressure-induced
connective tissue synthesis in pulmonary artery segments is
dependent on intact endothelium. J Clin Invest 1989; 84:
1005–1012
5 Thompson BT, Hassoun PM, Kradin RL, et al. Acute and
chronic hypoxic pulmonary hypertension in guinea pigs.
J Appl Physiol 1989; 66:920 –928
6 Bochnowicz S, Osborn RR, Hay DW, et al. Hypoxia-induced
pulmonary hypertension in an optimized environment for the
guinea pig. Lab Anim 1997; 31:347–356
7 Hassoun PM, Thompson BT, Steigman D, et al. Effect of
1904
Downloaded From: http://journal.publications.chestnet.org/ on 10/15/2014
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
heparin and warfarin on chronic hypoxic pulmonary hypertension and vascular remodeling in the guinea pig. Am Rev
Respir Dis 1989; 139:763–768
Castellot JJ Jr, Hoover RL, Karnovsky MJ. Glomerular endothelial cells secrete a heparin like inhibitor and a peptide
stimulator of mesangial cell proliferation. Am J Pathol 1986;
125:493–500
Thompson BT, Spence CR, Janssens SP, et al. Inhibition of
hypoxic pulmonary hypertension by heparins of differing in
vitro antiproliferative potency. Am J Respir Crit Care Med
1994; 149:1512–1517
Cindhuchao N, Quinn DA, Garg HG, et al. Heparin inhibits
SMC growth in the presence of human and fetal bovine
serum. Biochem Biophys Res Commun 2003; 302:84 – 88
Yu L, Quinn DA, Garg HG, et al. Cyclin-dependent kinase
inhibitor p27Kip1, but not p21WAF1/Cip1, is required for
inhibition of hypoxia-induced pulmonary hypertension and
remodeling by heparin in mice. Circ Res 2005; 97:937–945
Linhardt RJ, Gunay NS. Production and chemical processing
of low molecular weight heparins. Semin Thromb Haemost
1999; 25(suppl 3):5–16
Darien BJ, Fareed J, Centgraf KS, et al. Low molecular
weight heparin prevents the pulmonary hemodynamic and
pathomorphologic effects of endotoxin in a porcine acute lung
injury model. Shock 1998; 9:274 –281
Fareed J, Jeske W, Hoppensteadt D, et al. Are the available
low-molecular-weight heparin preparations the same? Semin
Thromb Haemost 1996; 22(suppl 1):77–91
Brieger D, Dawes J. Production method affects the pharmacokinetic and ex vivo biological properties of low molecular
weight heparins. Thromb Haemost 1997; 77:317–322
Hunter C, Barer GR, Shaw JW, et al. Growth of the heart and
lungs in hypoxic rodents: a model of human hypoxic disease.
Clin Sci Mol Med 1974; 46:375–391
Fulton RM, Hutchinson EC, Jones AM. Ventricular weight in
cardiac hypertrophy. Br Heart J 1952; 14:413– 420
Reid LM. The pulmonary circulation: remodeling in growth
and disease: the 1978 J. Burns Amberson lecture. Am Rev
Respir Dis 1979; 119:531–546
Meyrick B, Reid L. The effect of continued hypoxia on rat
pulmonary arterial c1irculation: an ultrastructural study. Lab
Invest 1978; 38:188 –200
Dahlberg CG, Thompson BT, Joseph PM, et al. Differential
effect of three commercial heparins on Na⫹/H⫹ exchange
and growth of PASMC. Am J Physiol 1996; 270:L260 –L265
Castellot JJ Jr, Addonizio ML, Rosenberg R, et al. Cultured
endothelial cells produce a heparin-like inhibitor of smooth
muscle cell growth. J Cell Biol 1981; 90:372–379
Clowes AW, Karnowsky MJ. Suppression by heparin of
smooth muscle cell proliferation in injured arteries. Nature
1977; 265:625– 626
Hoover RL, Rosenberg R, Haering W, et al. Inhibition of rat
arterial smooth muscle cell proliferation by heparin: II. In
vitro studies. Circ Res 1980; 47:578 –583
Benitz WE, Kelley RT, Anderson CM, et al. Endothelial heparan sulfate proteoglycan: I. Inhibitory effects on smooth muscle
cell proliferation. Am J Respir Cell Mol Biol 1990; 2:13–24
Castellot JJ Jr, Wong K, Herman B, et al. Binding and
internalization of heparin by vascular smooth muscle cells.
J Cell Physiol 1985; 124:13–20
Castellot JJ Jr, Favreau LV, Karnovsky MJ, et al. Inhibition of
vascular smooth muscle cell growth by endothelial cellderived heparin: possible role of a platelet endoglycosidase.
J Biol Chem 1982; 257:11256 –11260
Hales CA, Kradin RL, Brandstetter RD, et al. Impairment of
hypoxic pulmonary artery remodeling by heparin in mice. Am
Rev Respir Dis 1983; 128:747–751
Original Research
28 Garg HG, Roughley PJ, Hales CA. Proteoglycans in lung
disease. In: Garg HG, Hales CA, eds. Heparin as a potential
therapeutic agent to reverse vascular remodeling. New York,
NY: Marcel Dekker, 2003; 377–398
29 Cindhuchao N, Quinn DA, Garg HG, et al. Heparin inhibits
SMC growth in the presence of human and fetal bovine
serum. Biochem Biophys Res Commun 2003; 302:84 – 88
30 Hirsh J, Anand SS, Halperin JL, et al. Guide to anticoagulant
therapy: heparin; a statement for healthcare professionals from
the American Heart Association. Circulation 2001; 103:2994 –
3018
31 Gouin-Thibault I, Pautas E, Siguret V. Safety profile of
different low-molecular weight heparins used at therapeutic
dose. Drug Saf 2005; 28:333–349
32 Hirsh J, Levine MN. Low molecular weight heparin. Blood
1992; 79:1–17
33 Weitz JI. Low-molecular-weight heparins. N Engl J Med
1997; 337:688 – 698
34 Janssens SP, Thompson BT, Spence CR, et al. Polycythemia
and vascular remodeling in chronic hypoxic pulmonary hypertension in guinea pigs. J Appl Physiol 1991; 71:2218 –2223
35 Khorana AA, Sahni A, Altland OD, et al. Heparin inhibition of
endothelial cell proliferation and organization is dependent
on molecular weight. Arterioscler Thromb Vasc Biol 2003;
23:2110 –2115
36 Wong J, Wang N, Miller JW, et al. Modulation of human
fibroblast activity by selected angiogenesis inhibitors. Exp
Eye Res 1994; 58:439 – 451
www.chestjournal.org
Downloaded From: http://journal.publications.chestnet.org/ on 10/15/2014
37 Joseph PA, Garg HG, Thompson BT, et al. Influence of
molecular weight, protein core and charge of native heparin
fractions on pulmonary artery smooth muscle cell proliferation. Biochem Biophys Res Commun 1997; 241:18 –23
38 Wright TC Jr, Castellot JJ Jr, Petitou M, et al. Structural
determinants of heparin’s growth inhibitory activity: interdependence of oligosaccharide size and charge. J Biol Chem
1989; 264:1534 –1542
39 Dietrich CP, Shinjo SK, Moraes FA, et al. Structural features
and bleeding activity of commercial low molecular weight
heparins: neutralization by ATP and protamine. Semin
Thromb Haemost 1999; 25(suppl 3):43–50
40 Benitz WE, Bernfield M. Endothelial cell proteoglycans:
possible mediators of vascular responses to injury. Am J
Respir Cell Mol Biol 1990; 2:407– 408
41 Tiozzo R, Cingi MR, Reggiani D, et al. Effect of the desulfation
of heparin on its anticoagulant and anti-proliferative activity.
Thromb Res 1993; 70:99 –106
42 Fletcher JP, Ao PY, Hawthorne WJ. Antiproliferative effects
of low molecular weight heparin. ANZ J Surg 2004; 74:793–
796
43 Hirsh J, Warkentin TE, Raschke R, et al. Heparin and
low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and
safety. Chest 1998; 114:489S–510S
44 Fareed J, Hoppensteadt D, Jeske W, et al. Low molecular
weight heparins: are they different? Can J Cardiol 1998;
14(suppl E):28E–34E
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