www.elsevier.com/locate/vph

Vascular Pharmacology 40 (2003) 167–174

Enoxaparin—a low molecular weight heparin, restores the altered vascular

reactivity of resistance arteries in aged and aged–diabetic hamsters

Adriana Georgescua, Doina Popova,*, Monica Caprarub, Maya Simionescua

aInstitute of Cellular Biology and Pathology ‘‘N.Simionescu,’’ 8, B.P. Hasdeu Street, Bucharest 79691, Romaniab‘‘Dr. D. Gerota’’ Hospital, Bucharest, Romania

Received 1 December 2002; received in revised form 1 March 2003; accepted 1 April 2003

Abstract

We questioned whether the low molecular weight heparin enoxaparin acts upon the altered vascular reactivity of the resistance arteries in

normal biological aging and in aging associated with diabetes. Experiments were performed on isolated resistance arteries of young (4

months old), aged (16 months old), and aged–diabetic hamsters (16 months old and 5 months since streptozotocin injection), and the

reactivity was assessed by the myograph technique. The results showed that enoxaparin (60 mg/ml) had favorable effects on the vascular

reactivity in aged and aged–diabetic conditions, i.e., diminished the contractility of the arterial wall to 10�8–10�6 M noradrenaline (NA) and

to K+, and potentiated the impeded endothelium-dependent relaxation to 10�8–10�4 M acetylcholine (ACh). The effect was more

pronounced compared to that produced by unfractionated heparin (UFH). These pharmacological effects supplement the anticoagulant

properties of enoxaparin and may be of relevance for improving perfusion/circulation in the microvasculature of aged and of aged–diabetic

persons.

D 2003 Published by Elsevier Inc.

Keywords: Enoxaparin; Heparin; Diabetes

1. Introduction dose–response characteristics, a lower bleeding time (Cohen,

To date, the use of heparin in antithrombotic therapy is

limited due to the identification of several disadvantages.

Thus, the unfractionated heparin (UFH) binds to plasma

proteins (histidine-rich glycoproteins, vitronectin, lipopro-

teins, fibronectin, and fibrinogen), platelet factor 4, and high

molecular weight von Willebrand factor (secreted by plate-

lets and endothelial cells) that all contribute to a highly

variable anticoagulant response (Hirsh, 1998a). UFH has a

short plasma half life and a poor bioavailability (Hirsh,

1998b) and may increase the risk for thrombocytopenia

(Purcell and Fox, 1998) and osteoporosis (Muir et al., 1996).

These inconveniences were overcome by a new class of

anticoagulants, the low molecular weight heparins (LMWH)

that possess pharmacokinetic and biological advantages

over UFH (Hirsh et al., 1998; Waters and Azar, 1997;

Turpie, 2000). One of the LMWH is enoxaparin (Mr 4200

D), which exhibits a reduced nonspecific binding to plasma

proteins (Young et al., 1994), an improved predictability of

1537-1891/03/$ – see front matter D 2003 Published by Elsevier Inc.

doi:10.1016/S1537-1891(03)00041-7

* Corresponding author. Tel.: +40-21-411-0860; fax: +40-21-411-1143.

E-mail address: popov@simionescu.instcellbiopath.ro (D. Popov).

2000), and a reduced incidence of recurrent ischemic events

and need for revascularization therapy (O’Brien et al., 2000).

Enoxaparin was reported to be a safe anticoagulant during

pregnancy (Sanson et al., 1999), to prevent the occurrence of

thrombotic manifestations in chronic hemodialysed patients

(Reach et al., 1994), and an efficient treatment of vascular

pathologies such as the acute coronary syndromes (Ferguson,

2000), unstable angina, and the venous thromboembolism

(Hirsh, 1998c).

Previous investigations revealed that independent on

their anticoagulant properties, UFH inhibits the proliferation

of vascular smooth muscle cells (Wessel and Iberg, 1996;

Pukac et al., 1997; Mishra-Gorur and Castellot, 1999), has

blood pressure lowering effects in hypertension (Wilson et

al., 1981; Vasdev et al., 1994), enhances endothelial per-

meability (Guretzki et al., 1994), is an endothelium-depend-

ent venodilator in humans (Tangphao et al., 1999), improves

perfusion of microvessels in pancreatitis (Dobosz et al.,

1997), causes nitric oxide (NO)-mediated vasodilation of

coronary arterioles (Tiefenbacher and Chilian, 1997), and

prevents endothelial dysfunction associated with ischemia–

reperfusion injury (Kouretas et al., 1998) and diabetic

nephropathy (Solini et al., 1997).

A. Georgescu et al. / Vascular Pharmacology 40 (2003) 167–174168

Based on the data that report the effect of UFH in various

pathologies of the blood vessels, we questioned whether

enoxaparin has an effect on the resistance arteries (the

vascular segment responsible for the regulation of systemic

blood pressure), the reactivity of which is altered during

aging and at a faster rate in diabetes. We report here that in

aged and aged–diabetic hamsters, enoxaparin reduces the

contraction of the resistance arteries to noradrenaline (NA),

lowers the tension developed in K+, and increases the

relaxation to acetylcholine (ACh). In similar experimental

conditions, UFH does not affect the vessel contractility and

manifested rather modest improvements of the endothelium-

dependent relaxation.

2. Materials and methods

2.1. Reagents

NA, ACh, sodium nitroprusside (SNP), heparin, NG-

nitro-L-arginine methyl ester (L-NAME), streptozotocin,

HEPES, and the enzymatic kits for glucose and cholesterol

assays were purchased from Sigma (St. Louis, MO, USA).

Enoxaparin (clexane) was from Rhone-Poulenc Rorer (Neu-

illy sur Seine, France). All other reagents used were of

analytical grade.

2.2. Animals

Forty five male golden Syrian hamsters were divided into

three experimental groups: (i) aged animals (normal; 16

months old); (ii) aged–diabetic animals (16 months old) that

were induced diabetics 5 months earlier by administration of

a unique dose of 50 mg/kg bw streptozotocin solved in 50

mM citrate buffer pH 4.5; and (iii) young hamsters (normal),

4 months old used as controls.

2.3. Biochemical assays

For all experimental groups, the hamsters were slightly

anesthetized by an intraperitoneal injection of 5% chloral-

hydrate (0.05 ml/100 g bw) and blood was collected from

the venous retroorbital plexus on 2.7 mM EDTA under

sterile conditions. In separated plasma, the glucose and

cholesterol concentrations were enzymatically assayed.

2.4. Vascular reactivity of the resistance arteries

Hamsters were sacrificed by cervical dislocation, and

after laparotomy the small intestine (7–10 cm from the

pylorus) was excised and pinned down on Sylgaard resin in

a Petri dish. Segments (1–2 mm long) of the third order

branches of the mesenteric artery (resistance arteries) were

dissected out, threaded through two stainless steel wires (Ø:

40 mm), and mounted in a small vessel myograph (Model

410A, J.P. Trading, Denmark) as described by Mulvany and

Halpern (1997). The myograph chamber was filled with

HEPES salt solution (HPSS) containing (mM): HEPES (5),

NaCl (140), KCl (4.6), MgSO4 (1.17), CaCl2 (2.5), and

glucose (10) (Chulia et al., 1995; Thurston et al., 1995),

maintained at 37 �C, and continuously gassed with O2. After

an equilibration period of 20 min, the arteries were set to the

normalized internal circumference (0.9 times the circumfer-

ence they would have when relaxed and subjected to a

transmural pressure of 100 mm Hg). Subsequently, each

artery was subjected to a standard start procedure that

ensures the maximum isometric response. The procedure

consisted in the following steps (3 min each): (i) contraction

in 10�6 M NA in K-HPSS (HPSS supplemented with

equimolar concentrations of K instead of Na), (ii) wash in

HPSS, (iii) contraction in 10�6 M NA in HPSS, (iv) wash in

HPSS, and (v) step (i) repeated. All vascular reactivity

experiments were performed on resistance arteries with

mean internal diameter 250–290 mm (at 100 mm Hg).

2.4.1. Selection of the concentration of enoxaparin for in

vitro studies

In order to choose the enoxaparin concentration that may

cause either contraction or relaxation of the vascular wall in

vitro, a preliminary experiment was conducted as follows.

Isolated mesenteric resistance arteries (of either normal or

aged hamsters) maintained in basal conditions (HPSS) were

exposed for 10 min to various concentrations of enoxaparin

(10, 12, 14, 16, 18, 20 and 60 mg/ml). At all concentrations

tested, the force measured at the interface of the myograph

was �3.15 mN, except for 60 mg/ml (14.30 nM/ml) enox-

aparin that persistently decreased the force to �2.98 mN,

indicative for a slight vasorelaxing effect. The latter con-

centration of enoxaparin was employed for the assays of the

vascular reactivity in the presence of agonists (vasoconstric-

tors and vasodilators).

2.4.2. Effect of enoxaparin on the contraction of the

resistance arteries

After the routine standard start procedure, the resistance

arteries of young, aged, and aged–diabetic hamsters were

exposed to increasing concentrations of 10�8–10�4 M NA

cumulatively added to the organ bath, and the contractile

force recorded (in mN) at the interface of the apparatus. To

check for the effect of enoxaparin on the contraction to NA,

the arteries were preincubated for 10 min at 37 �C in 60 mg/ml enoxaparin in HPSS (in the organ bath of the myograph),

increasing concentrations of 10�8–10�4 M NAwere added,

and the contractile tension recorded (every 2 min).

To assay the contractile response to K+, the resistance

arteries were subjected to a discontinuous gradient of

increasing concentrations of K+, i.e., 24.4, 42.46, 64.10,

83.93, and 123.7 mM, and the force recorded. To check for

the effect of enoxaparin on the contraction to K+, the arteries

were preincubated for 10 min at 37 �C in 60 mg/ml

enoxaparin in HPSS and then exposed to the discontinuous

gradient of K+ (as above).

Pharmacology 40 (2003) 167–174 169

2.4.3. Effect of enoxaparin on the relaxation of the

resistance arteries

To measure the endothelium-dependent relaxation, the

resistance arteries (brought to the maximum isometric

response, as in 2.4) were precontracted in 3�10�6 M NA,

exposed to cumulative concentrations of 10�8–10�4 M

ACh, and the force recorded at 2-min intervals. The relaxa-

tion of the vascular wall was expressed as the percentage

from the NA-induced contraction of the artery.

To assay the effect of enoxaparin on endothelium-

dependent relaxation, arteries were precontracted in

3�10�6 M NA, incubated for 10 min with enoxaparin

(60, 120, or 240 mg/ml), and then increasing doses of

ACh (10�8–10�4 M) were added to the organ bath. Similar

experiments were conducted with 60 mg/ml UFH instead of

enoxaparin with the intent to compare their effects on

endothelium-dependent relaxation.

To test the role of NO in vasodilation of resistance

arteries, the activity of nitric oxide synthase (NOS) was

blocked by incubation of NA precontracted vessels in 10�4

M L-NAME for 10 min, and then the reactivity to 10�4 M

ACh in the absence or the presence of 60 mg/ml enoxaparin

was measured.

To assay the endothelium-independent relaxation, the

resistance arteries were precontracted in 3�10�6 M NA,

incubated for 10 min in 60 mg/ml either enoxaparin or UFH,

and the reactivity to cumulative doses of 10�8–10�4 M

SNP measured at 2 min intervals.

2.5. Data analysis

Data were expressed as mean±S.E.M. Tension developed

in NA and K+ was expressed as active wall tension (mN

mm�1 artery length)±S.E.M. and relaxation to ACh and

SNP was given as percentage from contraction to NA. The

concentration–response curves were compared by one-way

ANOVA. The data were considered significant when P<.05.

A. Georgescu et al. / Vascular

Fig. 1. The effect of enoxaparin on the contractility of the resistance arteries.

(a) The dose– response curves of the mesenteric arteries from aged (16

months old) hamsters exposed to 10�8–10�4 M NA in the presence or

absence of 60 mg/ml enoxaparin. (b) The contractile response to 10�6 M NA

3. Results

3.1. Plasma glucose and cholesterol concentrations

As shown in Table 1, in young hamsters (4 months of

biological age), the plasma glucose and cholesterol concen-

trations had values of 85±20 and 80±8 mg/dl, respectively.

Table 1

Plasma glucose and cholesterol concentrations for the three experimental

groups

Hamsters Glucose

(mg/dl)

Cholesterol

(mg/dl)

Young (4 months old) 85±20 80±8

Aged (16 months old) 149±27 125±19

Aged diabetics (16 months old) 290±15 245±12

in the presence or absence of 60 mg/ml enoxaparin: arteries from young (4

months old), aged (16 months old), and aged–diabetic hamsters (16 months

old, 5 months since streptozotocin injection). Note that enoxaparin reduces

the contractile response to 10�6 M NA in aged and aged–diabetic hamsters

and had no effect on the contractility of the arteries explanted from young

animals. (c) The response of the arteries explanted from aged hamsters to a

gradient of K+ in the presence or absence of 60 mg/ml enoxaparin. Note that

the drug reduced the tension developed by the vessel wall particularly up to

64.1 mM K+. The active wall tension is expressed as milliNewtons per

millimeter artery length. The data were obtained from nine animals for each

group; and for each mesenteric bed, two resistance arteries were

independently assayed.

A. Georgescu et al. / Vascular Pharmacology 40 (2003) 167–174170

In aged animals (16 months old), an �1.75-fold increase in

circulating glucose and �1.56-fold enhance in cholesterol

concentrations were detected. Addition of the diabetic

condition to the aged animals conducted to the doubling

of both glucose and cholesterol concentration in the plasma

comparatively with the figures obtained for normal aged

group. When related to the young hamster group, in aged–

diabetics, the plasma glucose and cholesterol were �3.4-

and �3-fold increased, respectively (Table 1).

3.2. Effects of enoxaparin on the contractility of resistance

arteries

As expected, in young, aged, and aged–diabetic ham-

sters, a dose-dependent contractile response of resistance

arteries to increasing concentrations of NA (10�8–10�4 M)

was recorded. Prior exposure (for 10 min) to 60 mg/ml

Fig. 2. The endothelium-dependent relaxation of resistance arteries to various conc

the organ bath. (a) Arteries explanted from young normal hamsters respond in a do

to 10�7–10�5 M ACh; whereas in the same conditions, heparin reduces the relaxat

(b) Vasorelaxation to ACh of the arteries of normal aged hamsters: The vessel’s rela

10�7 M ACh), whereas no effect on relaxation is detected at similar concentration o

in aged–diabetic group. However, addition of enoxaparin significantly improves th

the same concentration, heparin had no effect on relaxation. (d) Effects of various

aged hamsters. Note that the most potent relaxing effect of enoxaparin on ACh-d

enoxaparin, followed by supplementation of the organ bath

with 10�8–10�4 M NA, did not modify the dose–response

curve of the arteries from young hamsters (data not shown).

In contradistinction, the arteries of aged hamsters exposed to

60 mg/ml enoxaparin exhibited a significantly reduced

contractile tension when subjected to NA at concentrations

of 10�8–10�6 M as compared to the response in the absence

of enoxaparin (Fig. 1a). At higher NA concentrations

(3�10�6–10�4 M), enoxaparin did not modify the contract-

ile tension (Fig. 1a). Based on these data, we selected (i) for

precontraction of arteries, the concentration of 3�10�6 M

NA (situated at the plateau zone of the dose–response

curves, where the contractile response was not influenced

by enoxaparin), and (ii) for the evaluation of the force

developed by the arteries in all experimental groups, the

concentration of 10�6 M NA (were enoxaparin reduced

contractile force and showed favorable effects).

entration of ACh in the absence or presence enoxaparin or heparin added to

se-dependent manner to ACh. Addition of enoxaparin enhances vasodilation

ion to ACh; at 3�10�5–10�4 M ACh, enoxaparin or heparin have no effect.

xation is significantly enhanced in the presence of enoxaparin (starting from

f heparin. (c) The vasorelaxation to ACh is generally reduced in the arteries

e vessel’s relaxation to ACh mostly in the range of 10�7 to 10�5 M ACh; at

concentration of enoxaparin on relaxation to ACh assayed for the arteries of

ependent relaxation was obtained at low (60 mg/ml) concentration.

Fig. 3. Relaxation to ACh (10�5 M); effects of blocking NOS with L-

NAME (10�4 M) in the presence and absence of 60 mg/ml enoxaparin. The

responses of the resistance arteries from young, aged, and aged–diabetic

hamsters were comparatively examined. Nine animals were used for each

experimental group.

A. Georgescu et al. / Vascular Pharmacology 40 (2003) 167–174 171

As shown in Fig. 1b arteries of young hamsters

responded to 10�6 M NA with a tension of 1.59±0.18

mN/mm, a value that was similar to that obtained in the

presence of 60 mg/ml enoxaparin (1.50±0.18 mN/mm).

Arteries in aged group contracted in 10�6 M NA with a

tension of 3.22±0.15 mN/mm, i.e., twofold stronger than the

vessels from young animals; in the presence of 60 mg/ml

enoxaparin, the tension was reduced by half (1.56±0.17 mN/

mm) (Fig. 1b). The arteries from aged–diabetic group

developed in 10�6 M NA, a tension of 3.90±0.16 mN/mm

(�2.45-fold augmented vs. that recorded in the young

group); in the presence of 60 mg/ml enoxaparin, the tension

was reduced �1.34-fold (2.90±0.14 mN/mm) (Fig. 1b).

In another series of experiments, we tested the effect of

enoxaparin on the contractile response to various concen-

trations of K+ (known to modify the membrane potential).

The results showed that in young, aged, and aged–diabetic

hamsters, K+ concentrations from 24.4 to 64.1 mM induced

a gradual enhancement of the contractile response of the

resistance arteries. For the vessels of elder hamsters, the

highest tension (3.25±0.26 mN/mm) was recorded at 64.1

mM K+; above the latter concentration, a slight diminish-

ment in the contractile response of the vascular wall was

measured (Fig. 1c). Exposure of vessels to 60 mg/ml

enoxaparin reduced the contractility to K+, an effect that

diminished in parallel with the increase in K+ concentration.

In all experimental groups a particular reduced effect of

enoxaparin was found at concentrations of K+ over 64.1

mM (Fig. 1c).

3.3. Effect of UFH on the contractility of the resistance

arteries in aged hamsters

For the group of aged hamsters, the contractile force

developed by the resistance arteries exposed to 10�6 M NA

was 3.22±0.15 mN/mm. This value was only slightly

changed (3.04±0.20 mN/mm) in the presence of 60 mg/ml

UFH (a similar concentration that was used for enoxaparin).

In the same experimental group, the arteries subjected to 64.1

mM K+ in the absence or presence of 60 mg/ml UFH re-

sponded by a comparable contractile tension, i.e., 3.25±0.26

versus 3.31±0.24 mN/mm. These experiments indicated that

for isolated resistance arteries of aged hamsters UFH did not

change significantly either the contractile response to NA or

to K+.

3.4. Effects of enoxaparin on the endothelium-dependent

relaxation of the resistance arteries

For the three experimental groups, the resistance arteries

precontracted in NA (3�10�6 M) were exposed to increas-

ing concentrations (10�8–10�4 M) of the endothelium-

dependent vasodilator, ACh. For young animals, the relaxa-

tion of arteries attained a maximum of 85.59±4%; the

presence of 60 mg/ml enoxaparin increased vasodilation of

the arteries particularly for the interval of 10�7–10�5 M

ACh and had no effect at higher concentrations of ACh, i.e.,

3�10�5–10�4 M ACh (Fig. 2a). As expected, vessels of

aged hamsters had a reduced relaxation in ACh with a

maximum of 68.3±1.9%; enoxaparin (60 mg/ml) enhanced

significantly the endothelium-dependent relaxation in a

large interval (10�7–10�4 M) of ACh concentrations (Fig.

2b and d). The most reduced relaxation (46.58±2.7% from

precontraction) was observed for the arteries collected from

aged–diabetic hamsters (Fig. 2c); enoxaparin (60 mg/ml)

augmented the endothelium-dependent relaxation for the

interval of 10�7–10�5 M ACh (Fig. 2c). Comparatively,

at 10�5 M ACh, addition of 60 mg/ml enoxaparin in the

organ bath induced a slight enhancement (�8%) in endo-

thelium-dependent vasodilation of the arteries of young

group (Fig. 2a) and significant improvements of impeded

relaxation for the vessels in aged and aged–diabetics groups

(�37% and �22%, respectively) (Figs. 2b–d and 3).

In a separate experiment, we tested whether concentra-

tions of enoxaparin higher than 60 mg/ml would further

potentiate the endothelium–dependent relaxation. To this

intent, the resistance arteries in aged group were exposed for

10 min to either 60, 120, or 240 mg/ml enoxaparin, followed

by addition of increasing concentrations of ACh (10�8–

10�4 M). The results showed that 60 mg/ml enoxaparin

improved vasodilation in a large interval of ACh concen-

trations (Fig. 2b and d), while 120 and 240 mg/ml enoxaparin

had either no effect (at 10�8–10�5 M ACh) or induced

vasoconstriction (at 3�10�5–10�4 M ACh) (Fig. 2d).

To check whether NO is involved in the endothelium-

dependent relaxation, the activity of NOS was inhibited by

supplementing 10�4 M L-NAME to the organ bath of the

myograph. The results showed that relaxation in the pres-

ence of 10�4 M L-NAME and 10�5 M ACh measured

59.73±2.8 in young animals, 47.61±2.8 in aged, and

19.38±2% in aged–diabetic hamsters (Fig. 3). In 10�5 M

A. Georgescu et al. / Vascular Pharmacology 40 (2003) 167–174172

ACh supplemented with 10�4 M L-NAME and 60 mg/ml

enoxaparin, the relaxation was 71.42±1.9 in young animals,

59.01±1.9 in aged, and 24.37±1% in aged–diabetic ham-

sters (Fig. 3). The latter result suggests a less potent effect of

L-NAME in the presence of enoxaparin in the arteries of

young and aged hamsters; the data were not statistically

significant for the arteries of aged–diabetic animals (Fig. 3).

3.5. Effect of UFH on endothelium-dependent relaxation of

the resistance arteries

To find out whether heparin has a similar effect on

relaxation of the resistance arteries as enoxaparin, the

resistance arteries from young, aged, and aged–diabetic

groups were exposed for 10 min to 60 mg/ml heparin

(unfractionated), followed by addition of increasing con-

centrations of ACh (10�8–10�4 M). In the arteries of young

group, UFH had a rather reduced effect on ACh relaxation;

in contradistinction, within the same interval of ACh con-

centrations (10�8–10�5 M), 60 mg/ml enoxaparin improved

vasodilation (Fig. 2a). Experiments carried out on similar

arteries in both aged and aged–diabetic groups showed that

60 mg/ml UFH had no effect on ACh relaxation while a

similar concentration of enoxaparin stimulated vasodilation

(Fig. 2b and c).

3.6. Effects of enoxaparin and UFH on endothelium-

independent relaxation of the resistance arteries

The vasodilation of the resistance arteries to the endo-

thelium-independent donor SNP (10�8–10�4 M) was sim-

ilar for the young, aged, and aged–diabetic hamsters,

Fig. 4. A representative diagram of the endothelium-independent relaxation

of mesenteric resistance arteries explanted from young, aged, and aged–

diabetic hamsters in response to SNP supplemented with enoxaparin or

heparin, at 60 mg/ml. Similar diagrams were obtained for the three

experimental groups; neither enoxaparin nor heparin had any effect on

relaxation to 10�8–10�4 M SNP.

attaining a maximum of 78.68±3.7 mN/mm at the highest

concentration of the vasorelaxant (data not shown). Neither

enoxaparin nor UFH (60 mg/ml) has changed the relaxation

of arteries to SNP (Fig. 4).

4. Discussion

In this study, we questioned whether the LMWH enox-

aparin may influence the reactivity of resistance arteries to

vasoconstrictors and vasodilators, reportedly altered during

aging and diabetes. Experiments were performed in vitro on

the mesenteric resistance arteries from aged and aged–

diabetic animals (young normals used as control), a vascular

bed involved in the regulation of systemic blood pressure.

The golden Syrian hamster was elected as experimental

model due to several distinctive features: (i) its lipid

metabolism is similar to that of humans (Ohtani, 1990;

Sullivan et al., 1993); (ii) it has a propensity to develop

atherosclerotic lesions in response to high fat diet (Nistor et

al., 1987; Simionescu et al., 1996); and (iii) easily develops

type 1 diabetes upon injecting streptozotocin (Tomioka et

al., 1991; Popov and Simionescu, 1997).

We report that both normal biological aging and diabetes

induced in aged hamsters conduct to dysfunction of the

resistance arteries. Thus, the contraction to NA and to K+

was significantly higher than that of similar vessels

explanted from young animals, and the tension developed

by the vascular wall was the highest when aging was

associated with diabetes (Fig. 1). We assume that the

maximum contractile effect observed in �60 mM K+ is

due to the opening of voltage-gated and Ca2+-dependent

channels conducting to membrane depolarization and

increase in intracellular-free Ca2+ concentration ([Ca2+]i)

of smooth muscle cells. Above �60 mM K+, further

opening of these channels shifts the membrane potential to

equilibrium potential of K+ that stabilizes the smooth

muscle cells and causes relaxation (Zhao et al., 1997).

The endothelium-dependent relaxation of arteries in ACh

was found to be reduced in aged animals and particularly

diminished in aged–diabetic groups (Figs. 2 and 3), an

observation that corroborates well with the increased con-

tractile response and indicates a dysfunctional reactivity of

the cells of the vascular wall.

The concentration of enoxaparin elected for in vitro

testing of vascular reactivity was in the same range (mg/ml) with that used in cell culture conditions (16 mg/ml,

Manduteanu et al., 2002). When administered in vivo, the

doses of enoxaparin were 2�1.5 mg/kg bw in ischemic rats

(Stutzmann et al., 2002), 1.5 mg/kg bw for 4 days for obese

persons (Sanderink et al., 2002), and 2 mg/kg bw for 3 days

in heart transplant rejection (Schlaifer and Mills, 2000). One

can assume that in vivo bioavailability of enoxaparin to the

level of the microvasculature (small resistance arteries

included) will depend on its turnover in circulation as well

as on the route and frequency of administration.

A. Georgescu et al. / Vascular Pharmacology 40 (2003) 167–174 173

The main finding of this study is the favorable effect of

enoxaparin (60 mg/ml) on the vascular reactivity of resist-

ance arteries in aged and aged–diabetic conditions, i.e.,

diminishment of the contractile effect of 10�8–10�6 M NA

and of K+ (Fig. 1), and augmentation of the impeded

endothelium-dependent relaxation to ACh (Fig. 2). These

results corroborate well with those of Schlaifer and Mills

(2000) that reported significant improvements in micro-

vascular endothelium-dependent vasomotor function in

acute cardiac allograft rejection approximately 10 days after

treatment with enoxaparin.

Studies on expression of NOS are in progress in our

laboratory in order to define whether NOS or/and another

mediator trigger the effect of enoxaparin in young and aged

animals; from the data obtained, it seems that the NOS

pathway is less probable to be involved in aged–diabetic

hamsters.

The existent data for the effect of UFH are contradictory.

Administration of heparin in vivo in rats (250 units/day sc)

was reported to reduce the vasoconstriction to NA in

mesenteric arteries and to restore the vasodilator response

to ACh (Benchetrit et al., 1999). In dogs receiving 6 mg/kg

iv heparin, Kouretas et al. (1998) reported that heparin

preserved the vasoregulatory function of the endothelium

during brief episodes of ischemia–reperfusion injury in part

via the NO pathway. However, Bachetti et al. (1999)

showed that high-dose heparin administrated intravenously

in rats as a prolonged (4 h) therapeutic regimen has a

negative influence on the NO pathway impairing the endo-

thelium-dependent regulation of the vascular tone.

In our experiments, heparin, at the same concentration

and conditions as enoxaparin, has no effect on small arteries

contractility to either NA or K+ and the relaxation to ACh

was reduced as compared to enoxaparin. Recent reports

show that UFH and enoxaparin share several cellular effects

such as inhibition of nitrotyrosine formation (a hallmark of

vascular inflammation) in extracellular matrix proteins

(Baldus et al., 2001) and inhibition of activation of extrac-

ellular signal-regulated kinase in smooth muscle cells, a

process correlated with inhibition of mitogenesis (Bretsch-

neider and Schror, 2001). In addition, enoxaparin was

reported to inhibit the expression of heparanase produced

by the activated T-lymphocytes (Pacheco and Kerdel, 2001).

The beneficial properties of enoxaparin reported here

may prove relevant for the perfusion/circulation at the level

of the microvasculature of aged persons and old–diabetic

patients. The ability to reduce vasoconstriction and the

augmentation of the endothelium-dependent relaxation are

a novel attribute of enoxaparin besides those already

reported, such as the bioavailability, the efficiency in the

treatment of thromboembolic incidents and of the acute

coronary syndromes (Zed et al., 1999), inhibition of cell

adhesion molecule-dependent monocyte adhesion to endo-

thelial cells (Manduteanu et al., 2002), and the effect on the

rising of von Willebrand factor that complicate the unstable

coronary artery disease (Montalescot et al., 1998).

References

Bachetti, T., Pasini, E., Clini, E., Cremona, G., Ferrari, R., 1999. High-dose

heparin impairs nitric oxide pathway and vasomotion in rats. Circula-

tion 99, 2861–2863.

Baldus, S., Eiserich, J.P., Mani, A., Castro, L., Figueroa, M., Chumley, P.,

Ma, W.X., Tousson, A., White, C.R., Bullard, D.C., Brennan, M.L.,

Lusis, A.J., Moore, K.P., Freeman, B.A., 2001. Endothelial transcytosis

of myeloperoxidase confers specificity to vascular ECM proteins as

targets of tyrosine nitration. J. Clin. Invest. 108 (12), 1759–1770.

Benchetrit, S., Mandelbaum, A., Bernheim, J., Podjarny, E., Green, J., Katz,

B., Rathaus, M., 1999. Altered vascular reactivity following partial

nephrectomy in the rat: a possible mechanism of the blood-pressure-

lowering effect of heparin. Nephrol. Dial. Transplant 14, 64–69.

Bretschneider, E., Schror, K., 2001. Cellular effects of factor Xa on vascular

smooth muscle cells—inhibition by heparins? Semin. Thromb. Hemost.

27, 489–493.

Chulia, T., Gonzales, P., Del Rio, M., Tejerina, T., 1995. Comparative study

of elgodipine and nisoldipine on the contractile response of various

isolated blood vessels. Eur. J. Pharmacol. 285, 115–122.

Cohen, M., 2000. Initial experience with the low-molecular-weight heparin,

enoxaparin, in combination with the platelet glycoprotein IIb/IIIa block-

er, tirofiban, in patients with non-ST segment elevation acute coronary

syndromes. J. Invasive Cardiol. 12, E5–E9.

Dobosz, M., Hac, S., Mionskowska, L., Dobrowolski, S., Wajda, Z., 1997.

Microcirculatory disturbances of the pancreas in cerulein-induced acute

pancreatitis in rats with reference to L-arginine, heparin, and procaine

treatment. Pharmacol. Res. 36 (2), 123–128.

Ferguson, J.J., 2000. Combining low-molecular-weight heparin and glyco-

protein IIb/IIIa antagonists for the treatment of acute coronary syn-

dromes: the NICE 3 story. J. Invasive Cardiol. 12, E10–E13.

Guretzki, H.J., Schleicher, E., Gerbitz, K.D., Olgemoeller, B., 1994. Hep-

arin induces endothelial extracellular matrix alterations and barrier dys-

function. Am. J. Physiol. 267, C946–C954.

Hirsh, J., 1998a. Low-molecular-weight heparin for the treatment of venous

thromboembolism. Am. Heart J. 135, S336–S342.

Hirsh, J., 1998b. Recent clinical trials in the treatment of venous throm-

boembolism and unstable angina with low molecular weight heparins.

Curr. Opin. Hematol. 5, 360–365.

Hirsh, J., 1998c. Low-molecular-weight heparin. A review of the results of

recent studies of the treatment of venous thromboembolism and unsta-

ble angina. Circulation 98, 1575–1582.

Hirsh, J., Warkentin, Th.E., Raschke, R., Granger, C., Ohman, E.M., Dalen,

J.E., 1998. Heparin and low-molecular-weight heparin. Mechanisms of

action, pharmacokinetics, dosing considerations, monitoring, efficacy,

and safety. Chest 114, 489S–510S.

Kouretas, P.C., Kim, Y.D., Cahill, P.A., Myers, A.K., To, L.N., Wang,

Y.N., 1998. Heparin preserves nitric oxide activity in coronary endo-

thelium during ischemia-reperfusion injury. Ann. Thorac. Surg. 66,

1210–1215.

Manduteanu, I., Voinea, M., Capraru, M., Dragomir, E., Simionescu, M.,

2002. A novel attribute of enoxaparin: inhibition of monocyte adhesion

to endothelial cells by a mechanism involving cell adhesion molecules.

Pharmacology 65, 32–37.

Mishra-Gorur, K., Castellot, J.J., 1999. Heparin rapidly and selectively

regulates protein tyrosine phosphorylation in vascular smooth muscle

cells. J. Cell. Physiol. 178, 205–215.

Montalescot, G., Philippe, F., Ankri, A., Vicaut, E., Bearez, E., Poulard,

J.E., Carrie, D., Flammang, D., Dutoit, A., Carayon, A., Jardel, C.,

Chevrot, M., Bastard, J.P., Bigonzi, F., Thomas, D., 1998. Early in-

crease of von Willebrand factor predicts adverse outcome in unstable

coronary artery disease. Beneficial effects of enoxaparin. Circulation

98, 294–299.

Muir, J.M., Andrew, M., Hirsh, J., Weitz, J.I., Young, E., Deschamps, P.,

Shaughnessy, S.G., 1996. Histomorphometric analysis of the effects of

standard heparin on trabecular bone in vivo. Blood 88, 1314–1320.

Mulvany, M.J., Halpern, W., 1997. Contractile properties of small arteriolar

A. Georgescu et al. / Vascular Pharmacology 40 (2003) 167–174174

resistance vessels in spontaneously hypertensive and normotensive rats.

Circ. Res. 41, 19–26.

Nistor, A., Bulla, A.A., Filip, D.A., Radu, A., 1987. The hyperlipidemic

hamster as a model of experimental atherosclerosis. Atherosclerosis 68,

159–173.

O’Brien, B.J., Willan, A., Blackhouse, G., Goeree, R., Cohen, M., Good-

man, S., 2000. Will the use of low-molecular-weight heparin (enoxa-

parin) in patients with acute coronary syndrome save costs in Canada?

Am. Heart J. 139, 423–429.

Ohtani, H., 1990. Effects of dietary cholesterol and fatty acids on plasma

cholesterol and hepatic lipoprotein metabolism. J. Lipid Res. 31,

1413–1422.

Pacheco, H., Kerdel, F., 2001. Successful treatment of lichen planus with

low-molecular-weight heparin: a case series of seven patients. J. Derm.

Treat. 12, 123–126.

Popov, D., Simionescu, M., 1997. Alterations of lung structure in exper-

imental diabetes, and diabetes associated with hyperlipidemia in ham-

sters. Eur. Respir. J. 10, 1850–1858.

Pukac, L.A., Carter, J.E., Ottlinger, M.E., Karnovsky, M.J., 1997. Mecha-

nisms of inhibition by heparin of PDGF stimulated MAP kinase acti-

vation in vascular smooth muscle cells. J. Cell. Physiol. 172, 69–78.

Purcell, H., Fox, K.M., 1998. Current roles and further possibilities for low

molecular weight heparins in unstable angina. Eur. Heart J. 19 (Suppl. K),

K18–K23.

Reach, I., Thebaud, H.E., Dupuy, C.A., Kuntziger, H., Debure, A., Fievet,

P., Thoumazou, G., de Groc, F., Teboulle, D., Combe, S., 1994. Opti-

mization of enoxaparin dose in the prevention of coagulation in the

circuits hemodialysis: results of a multicenter study. Nephrologie 15,

395–401.

Sanderink, G.J., Guimart, C.G., Ozoux, M.L., Jarivalla, N.U., Shukla, U.A.,

Boutouyrie, B.X., 2002. Pharmacokinetics and pharmacodynamics of

the prophylactic dose of enoxaparin once daily over 4 days in patients

with renal impairment. Thromb. Res. 105, 225–231.

Sanson, B.J., Lensing, A.W.A., Prins, M.H., Ginsberg, J.S., Barkagan, Z.S.,

Lavenne-Pardonge, E., Brenner, B., Dulitzky, M., Nielsen, J.D., Boda,

Z., Turi, S., Mac Gillavry, M.R., Hamulyak, K., Theunissen, I.M., Hunt,

B.J., Buller, H.R., 1999. Safety of low-molecular-weight heparin in

pregnancy: a systematic review. Thromb. Haemost. 81, 668–672.

Schlaifer, J.D., Mills, R.M., 2000. Effects of low molecular weight heparin

on coronary endothelial function in acute cellular heart transplant re-

jection. Am. J. Cardiol. 86, 117–120.

Simionescu, M., Popov, D., Sima, A., Hasu, M., Costache, G., Faitar, S.,

Vulpanovici, A., Stancu, C., Stern, D., Simionescu, N., 1996. Pathobio-

chemistry of combined diabetes and atherosclerosis studied on a novel

animal model: the hyperlipemic hyperglycemic hamster. Am. J. Pathol.

148, 997–1014.

Solini, A., Vergnani, L., Ricci, F., Crepaldi, G., 1997. Glycosaminiglycans

delay the progression of nephropathy in NIDDM. Diabetes Care 20,

813–817.

Stutzmann, J.M., Mary, V., Wahl, F., Grosjean-Piot, O., Uzan, A., Pratt, J.,

2002. Neuroprotective profile of enoxaparin, a low molecular weight

heparin, in vivo models of cerebral ischemia or traumatic brain injury in

rats: a review. CNS Drug Rev. 8, 1–30.

Sullivan, M.P., Cerda, J.J., Robbins, F.L., Burgin, C.W., Beatty, R.J., 1993.

The gerbil, hamster and guinea pig as rodent models for hyperlipidemia.

Lab. Anim. Sci. 43, 575–578.

Tangphao, O., Chalon, S., Moreno, H.J., Abiose, A.K., Blaschke, T.F.,

Hoffman, B.B., 1999. Heparin-induced vasodilation in human hand

veins. Clin. Pharmacol. Ther. 66, 232–238.

Thurston, G., Baldwin, A.L., Wilson, L.M., 1995. Changes in endothelial

actin cytoskeleton at leakage sites in the rat mesenteric vasculature. Am.

J. Physiol. 266, H316–H329.

Tiefenbacher, C.P., Chilian, W.M., 1997. Basic fibroblast growth factor and

heparin influence coronary arteriolar tone by causing endothelium-de-

pendent dilation. Cardiovasc. Res. 34, 411–417.

Tomioka, T., Fujii, H., Hirota, M., Ueno, K., Pour, P.M., 1991. The patterns

of beta-cell regeneration in untreated diabetic and insulin-treated dia-

betic Syrian hamsters after streptozotocin treatment. Int. J. Pancreatol.

8, 355–366.

Turpie, A.G.G., 2000. Can we differentiate the low-molecular-weight hep-

arins? Clin. Cardiol. 23, I-4– I-7.

Vasdev, S., Ford, C.A., Longerich, L., Barret, B., Parai, S., Campbell, N.,

1994. Oral treatment with low molecular weight heparin normalizes

blood pressure in hypertensive rats. Artery 21, 1–28.

Waters, D.D., Azar, R.R., 1997. Low molecular-weight heparins for unsta-

ble angina: a better mousetrap? Circulation 96, 3–5.

Wessel, H.P., Iberg, N., 1996. The smooth muscle cell antiproliferative

activity of heparin sulfate model oligosaccharides. Bioorg. Med. Chem.

Lett. 6 (4), 427–430.

Wilson, S.K., Solez, K., Boitnott, J., Heptinstall, R.H., 1981. The effect of

heparin treatment of hypertension and vascular lesions in stroke prone

spontaneously hypertensive rats. Am. J. Pathol. 102, 62–71.

Young, E., Wells, P., Holloway, S., Weitz, J., Hirsh, J., 1994. Ex-vivo and

in-vitro evidence that low molecular weight heparins exhibit less bind-

ing to plasma proteins than UFH. Thromb. Haemost. 71, 300–304.

Zed, P.J., Tisdale, J.E., Borzak, S., 1999. Low-molecular-weight heparins in

the management of acute coronary syndromes. Arch. Intern. Med. 159,

1849–1857.

Zhao, Y.-J., Wang, J., Rubin, L.J., Yuan, X.J., 1997. Inhibition of Kv and

Kca channels antagonizes NO-induced relaxation in pulmonary artery.

Am. J. Physiol. 272, H904–H912.