Journal of Pharmacology, 222 (1992) 175-179 0 1992 Elsevier Science Publishers B.V. Ail rights reserved 0014-2999/92/$05.00

175

European

EJP 21140

Short communication

Modulation of vascular reactivity by vasoactive peptides in aortic rings from hypercholesterolemic rabbits Linda A. Merkel Department

of Cardiovascular

Pharmacology,

and Glenda E. Bilder

Rhc%e-Poulenc Rorer Central Research, PA 19426-0107, USA

500 Arcola

Road,

P.O. Box 1200, CoNegeville,

Received 5 August 1992, accepted 8 September 1992

The effect of moderate elevation in serum cholesterol on vascular reactivity to epidermal growth factor (EGF), endothelin (ET-l) and thrombin, vasoactive peptides present at sites of vascular injury, was examined in isolated aortic rings from rabbits fed either a casein-rich or a control diet for lo-12 weeks. In rings from hypercholesterolemic rabbits, development of maximal isometric tension to all peptide agonists was increased 22 f 0.6% while the EC,, for contraction was decreased. Vasorelaxant responses to nitroprusside, an endothelium-independent dilator, were largely intact, while those to A231897, an endothelium-dependent agent, were attenuated. These data suggest that elevation in serum cholesterol in the absence of atherosclerotic lesions is sufficient to increase vascular reactivity to peptide vasoactive mediators, an effect which may predispose arteries to vasospasm. EGF (epidermal growth factor); Endothelin; Thrombin; Hypercholesterolemia; Aorta (rabbit); Vasospasm; Endothelium

1. Introduction

released from activated platelets (Oka and Orth, 19831, endothelin

Clinical and experimental observations suggest that hypercholesterolemia and atherosclerosis enhance vascular reactivity and possibly predispose arteries to vasospasm (Hoak, 1988; Vanhoutte, 1990; Merkel et al., 1990). Although the mechanisms for vasospasm are incompletely understood, it is postulated that endotheha1 injury and platelet-derived factors are important

(Hoak, 1988; Vanhoutte, holesterolemia

1990). Specifically,

hyperc-

increases fragility of platelets (Carvulho

et al., 19741, diminishes receptor-mediated

endothe-

lium-dependent relaxing factor (EDRF) release to serotonin, thrombin and substance P (Hoak, 1988; Vanhoutte, 1990) and increases the direct smooth muscle vasoconstrictor effect of KCI, serotonin and

phenylephrine (Merkel et al., 1990; Galle et al., 1990). We previously reported that vascular reactivity to the low molecular weight agonists, serotonin, KC1 and iso-

proteienol

were differentially

elevation in serum cholesterol.

(ET-l),

a peptide

secreted by endothelial

and smooth muscle cells (Yanagisawa et al., 1988; Resink et al., 1990) and thrombin, a blood product generated

by the coagulation

cascade. These sub-

stances are potent vasoactive agonists (Yanagisawa et al., 1988; White et al., 1980; Berk and Alexander, 1989) in normocholesterolemic animals and are peptides most likely found at sites of vascular injury (Vanhoutte, 1990), either alone, collectively or consecutively, since for example thrombin can stimulate endothelin gene expression (Yanagisawa et al., 1988) and peptide release (Boulanger and Liischer, 1990). Our results show that a moderate increase in serum cholesterol potenti-

ates the vasoconstrictor activity of EGF, ET-1 and thrombin. Since we found a partially atenuated vasodilator response to the endothelium-dependent A23187, these effects can be attributed, at least in part, to impaired release of EDRF.

enhanced with modest In this communication

we present the effect of moderate elevations in serum cholesterol on the vasoconstrictor activities of three peptides: epidermal growth factor (EGF), a mitogen

Correspondence to: L.A. Merkel, NW4, RhBne-Poulenc Rorer Central Research, 500 Arcola Road, Collegeville, PA 19426-0107, USA. Tel. 1.215454 5086, fax 1.215.454 5658.

2. Materials

and methods

2.1. Animals

New Zealand white male rabbits of the SPF (special pathogen free) strain were studied at 18 weeks of age and were divided into two groups (N = 8 each) which received an experimental or a control diet for an

176

additional 10 weeks. The control group received standard lab chow, the experimental (hypercholesterolemic) group received a diet previously shown to elevate serum cholesterol (Wistar Diet No. 12203, Dyets, Bethlehem, PA, USA) containing (g/kg): casein 246; sucrose 398; corn oil 10; hydrogenated coconut oil 10 and nutrients.

10.0. The aorta was cleaned of fat, blood and adhering tissue, cut into rings approximately 2 mm wide, which were mounted into water-jacketed tissue baths (10 ml). Each ring segment was suspended on L-shaped hooks between a stainless steel rod and a force transducer (Grass, FT-03) and was equilibrated for 60 min at a resting tension of 2 g with buffer changes at 5, 10, 15, 30, 45 and 60 min. Rings were exposed to 36 mM KC1 and allowed to relax before contractions were elicited by the addition of agonists to the baths in a cumulative fashion. Each concentration of EGF and thrombin remained in the bath for 10 min or until a new steady state was reached. Each concentration of ET-l remained in the bath for 5 min or until a new steady state was reached. Tension development was expressed as percent of the KC1 response. When vasodilators

2.2. Isometric tension measurement

Rabbits were anesthetized (sodium pentobarbital, 80 mg/kg) and the abdominal aorta was excised quickly and placed into warm (37”C), oxygenated (95% 0,, 5% CO,) Krebs-Henseleit buffer (pH 7.4) of the following composition (mM); NaCl 118; KC1 4.7; CaCl, 2.5; MgSO, 1.2; KH,PO, 1.2; NaHCO, 25.0 and glucose

100

60 1 60 --

-9

-6

-7

EGF [LOG

-6

M]

125 -

l ----6

100--

-9

-6 ENDOTHELIN

[LOG

I

I

-7

-6

M]

80 -;ei 60 -!,:pT?

0

40 --

20--Ty/y o-

n 0

2

I 6

4

THROMBIN

0

I 10

[U/ml]

Fig. 1. Dose-response curves to EGF (top), ET-1 (middle) and thrombin (bottom) in rabbit aortic rings from normocholesterolemic (0) and hypercholesterolemic (0) rabbits. Rings were exposed to cumulative concentrations of either peptide agonist and isometric tension was measured. The abscissa shows ET-l (top) and EGF (middle) concentration in log molar units, while thrombin (bottom) is expressed as units/ml. The ordinate shows percent of isometric tension to 36 mM KCI. Data are expressed as meansfS.E.M. for eight different rabbits.

177

were examined, rings were precontracted with 3 PM prostaglandin Fza (PGF,,), before exposure to cumulative doses of sodium nitroprusside (SNP) or A23187.

cholesterol levels of hypercholesterolemic rabbits were elevated 2.5fold (80 f 21 vs. 31 f 3.5 mg/dl; P < 0.05). 3.2. Vascular reactivity

2.3. Serum cholesterol determination

Blood was withdrawn from the ear artery of each rabbit at the beginning of the study, after 1 month, and at the end of the study (after lo-12 weeks). Total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol in serum were determined using the LDL-Direct Plus assay kit from Isolab, Inc. (Akron, OH). The standard curve was constructed using bovine cholesterol. 2.4. Histology

At the time of killing, random sections of the abdominal aorta were preserved in 10% buffered formalin. The tissue was histologically processed, stained with hematoxylin-eosin and evaluated microscopically for presence of intimal hyperplasia, increased extracellular matrix and evidence of lipid deposition. 2.5. Statistics

Data are expressed as means f S.E.M. Means were compared using the non-paired Student’s t-test and differences were considered significant at P < 0.05. IC,, values were determined using a linear curve fitting program (RS/l, Bolt, Beranek and Newman, Boston, MA). 2.6. Materials

EGF (from mouse submaxillary glands) was purchased from Toyobo (New York, NY), ET-l was obtained from Cambridge Research Biochemicals (Valley Stream, NY). cy-Thrombin (from human plasma; > 99% free of other clotting factors), sodium nitroprusside, L-nitroarginine and A23187 were purchased from Sigma (St. Louis, MO, USA). 3. Results 3. I. Serum cholesterol

After lo-12 weeks on the Wistar diet, total serum cholesterol in the experimental group was elevated approximately 4.5-fold (P < 0.05) compared to control rabbits which had received regular chow (270 f 60 mg/dl; N = 8 vs. 61 f 12 mg/dl, N = 8, respectively). LDL cholesterol levels were elevated 6-fold in the hypercholesterolemic rabbits compared to control rabbits (80 f 23 vs. 13 f 3 mg/dl, respectively), while HDL

All three peptide agonists were potent vasoconstrictors which produced a sustained tonic contraction in both test groups; compared to control rabbits, maximal isometric tension in rings from hypercholesterolemic rabbits was increased 22 f 0.6%; in addition, the EC,, for contraction was markedly reduced in the diet group. With EGF as vasoconstrictor, isometric tension increased significantly from 42 f 8.8 to 64 _+7.6% of the KC1 response (N = 8, P < 0.05). The half-maximal concentration necessary for contraction decreased approximately 6-fold from 51 f 5.0 to 8.3 4 2.3 nM; P < 0.05) (fig. 1). The ET-l response, similar to EGF, was enhanced in rings from hypercholesterolemic rabbits (106 f 6 vs. 127 f 3% of KCI-induced tension). ET-l was a potent vasoconstrictor with an EC,, value of 14.8 + 2.6 and 9.7 f 1.8 nM in control and hypercholesterolemic rings, respectively. The contractile response to thrombin was 60 + 14 and 83 _+6% of KCI-induced tension in control and hypercholesterolemic rabbits, respectively. The thrombin concentration necessary for halfmaximal contraction was significantly lower in the diet group compared to controls (0.99 k 0.08 vs. 1.8 f 0.34 U/ml, respectively). 3.3. Endothelium-dependent

responses

In order to assess the functional integrity of the endothelium, dose responses to the three peptide agonists were also examined in the presence of Lnitroarginine (L-Noarg), an inhibitor of nitric oxide release. In rings from control rabbits, pretreatment with 30 PM L-Noarg for 20 min prior to the cumulative dose response to EGF, ET-l or thrombin, resulted in an increase in maximal isometric tension of approximately 20%. This phenomenon was not observed in rings from hypercholesterolemic animals. On the same note, we examined the vasodilator response to an endothelium-dependent agent, the calcium ionophore A23187 and compared it with the endothelium-independent agent SNP. In rings from control rabbits we observed 71+ 9.1 and 95 rf: 4.6% relaxation of PGF,,-induced tension with the highest concentration of SNP and A23187, respectively. In rings from the diet group, relaxation to SNP was largely conserved (58 + 9.5%), while the response to the calcium ionophore was impaired (50 f 6.8%) (fig. 2). 3.4. Histology

The overt presence of atherosclerosis, such as intima1 hyperplasia, increased extracellular matrix and lipid

O-l -9

-7

-0

-6

I -5

-6

-5

SNP [LOG M]

“ig

-7

-0 A23187

[LOG M]

Fig. 2. Dose-response curve to SNP (top) and A23187 (bottom) in rings from control (0) and hypercholesterolemic (0) animals precontracted with PGF,,.

deposition was not detected in cross sections of the thoracic or abdominal aorta from hypercholesterolemic rabbits. The sections from hypercholesterolemic animals appeared completely normal and were virtually indistinguishable from those of the control rabbits (data not shown). 4. Discussion Recently we reported that moderate elevation of serum cholesterol in rabbits was sufficient to modulate vascular reactivity to serotonin, KC1 and isoproterenol (Merkel et al., 1990). We now show that with comparable serum cholesterol levels, the vasoconstrictor activity of the endogenous peptide agonists EGF, ET-1 and thrombin are also enhanced. While we observed a 22% increase in maximal isometric tension in hypercholesterolemic animals with all three agonists, the shift in EC,, values was most pronounced (6-fold) with EGF, a mitogen, whose receptor belongs to the tyrosine kinase family. Even though EGF concentrations used here are markedly higher than in human plasma (Oka and Orth, 19831, higher local EGF concentrations might be anticipated at sites. in the arterial wall following platelet adherence and degranulation. To our knowledge the effect of hypercholesterolemia on vasoreactivity of ET1, one of the most potent vasoconstrictors known

(Yanagisawa et al., 1988), has not been previously examined. It is emphasized that while the diet-induced elevation in serum cholesterol in these animals approximate that reported for atherosclerotic conditions in man (Wayne et al., 1981>, this study was of short duration (lo-12 weeks) and altered reactivity to ET-l occurred without gross atherosclerotic changes in vascular pathology. Moderate hypercholesterolemia also influenced specific aspects of vasoconstriction induced by thrombin. Although marked hypercholesterolemia and severe atherosclerosis are known to potentiate thrombin-dependent vasoactivity (Freiman et al., 19861, our results suggest that vascular reactivity to thrombin occurs with a lesser degree of hypercholesterolemia and in the absence of atherosclerosis. We speculate that possible mechanisms by which elevated serum cholesterol or lipoprotein fractions potentiate the vasoconstrictor actions of EGF, ET-l and thrombin include endothelium-dependent pathways as well as direct effects on smooth muscle by modulation of intracellular calcium. Endothelium-dependent modulation of thrombin induced vasoconstriction has been established (Hoak, 1988; Vanhoutte, 1990) and thrombin-induced EDRF release is depressed in atherosclerosis (Freiman et al., 1986). Whether ET-l-induced vasoconstriction is endothelium-dependent is controversial and the modulatory role of EDRF on EGF-induced contractions is unknown, even though it seems possible that hypercholesterolemia may influence endothelium-independent vasoconstrictor effects. Our data using L-Noarg, an inhibitor of EDRF release, demonstrate an increase in maximal isometric tension in rings from control, but not hypercholesterolemic animals, indicating that under normal conditions the vasoconstrictor actions of all three peptide agonists are modulated by EDRF. Under conditions of elevated serum lipids, however, this influence of EDRF has disappeared, suggesting that the function of the endothelium is impaired. These findings are supported by the attenuated vasodilator response to the calcium ionophore A23187, an endothelium-dependent agent. However, vasodilation. to A23187 was only partially inhibited, arguing that other direct effects on smooth muscle may be responsible. ET-l and thrombin have been shown to elevate intracellular calcium in vascular smooth muscle via activation of phospholipase C, resulting in the production of the. two second messengers inositol-1,4,5-triphosphate and diacylglycerol and subsequent increase in intracellular calcium (Ohlstein et al., 1989). It can be envisioned that serum cholesterol might influence intracellular calcium mobilization or calcium fluxes, thereby sensitizing smooth muscle to the vasoconstrictor actions of these peptide agonists. The observation that in cultured vascular smooth muscle cells low concentrations of LDL (7-10 pg protein/ml) increase

179

intracellular calcium and induce a biphasic change in intracellular pH by an LPL receptor-independent mechanism (Sachinites et al., 1990) and increase phosphoinositide turnover by an LDL receptor-dependent mechanism (Block et al., 1988>, suggests potential cellular sites for interaction. In vitro low concentrations of oxidized LDL, but not native LDL, act directly on smooth muscle to enhance vasoconstriction induced by norepinephrine, phenylephrine, serotonin and KC1 (Galle et al., 1990). Acknowledgement Sincere appreciation is extended to Drs. Mark H. Perrone and P. Macke Consigny for their helpful criticism of the manuscript. The authors are grateful to Ms. Luz Rivera, Mr. Dennis Colussi and Mr. Charles Kasiewski for their expert technical skills and to Drs. Thomas Hodge and Marion Valerio for the histological support.

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