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(Circulation. 1996;94:2703-2707.)
© 1996 American Heart Association, Inc.

Articles

Effects of Low-Dose Heparin Infusion on Arterial Endothelin-1 Release in Humans

P.M. Piatti, MD; L.D. Monti, MD; G. Valsecchi, MD; M. Conti, MD; R. Nasser, MD; B. Guazzini, MD; E. Fochesato, MD; C.V. Phan; A.E. Pontiroli, MD; G. Pozza, MD

the Istituto Scientifico H. San Raffaele, Cattedra di Clinica Medica, University of Milan, Italy.

Correspondence to PierMarco Piatti, MD, Istituto Scientifico H. San Raffaele, Via Olgettina 60, 20132 Milano, Italy.

	Abstract

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Background The aim of this study was to evaluate the effect of low-dose heparin infusion on arterialized endothelin-1 (ET-1) release in the presence of fasting or high insulin levels in healthy humans.

Methods and Results Eleven normal subjects underwent two tests in random order lasting 240 minutes. A primed (250 IU), continuous heparin (600 IU/h) infusion was performed in test 1; saline was infused in test 2 as control. At 120 minutes, a euglycemic hyperinsulinemic clamp (25 mU·kg^-1·h^-1) was started that lasted 2 hours in both tests. Two hours after heparin infusion (test 1), ET-1 levels decreased by 32% (3.52±0.60 to 3.02±0.73 pg/mL), while nitric oxide (NO) and forearm blood flow increased by 29% and 14%, respectively. During saline infusion, ET-1, nitric oxide, and forearm blood flow remained unchanged. There was a significant interaction between the effect of decreasing ET-1 levels and the heparin treatment (F, 4.06; df, 3.30; P<.01). The decrease in ET-1 levels was significantly correlated with the increase in forearm blood flow in test 1 (r=.74; P<.01) but not in test 2. During the heparin/insulin period, ET-1 increased by 25%, returning to fasting values; nitric oxide levels increased by 12%; and forearm blood flow remained unchanged.

Conclusions The present study showed that it is possible to decrease ET-1 levels by use of low-dose heparin infusion in humans. This effect seems mediated by a simultaneous increase in nitric oxide levels and is completely reversed by a mild increase in insulin concentrations.

Key Words: heparin • insulin • endothelium-derived factors

	Introduction

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Coronary vascular tone is regulated by the production both of vasoconstrictor substances such as ET-1^{1 2} and vasodilator substances such as NO.³ Recently, considerable attention has been attributed to the potential role of ET-1 in vascular disease, and increased concentrations of ET-1 were described in a variety of pathological conditions.^{4 5 6} Haynes and Webb⁷ found that a selective endothelin receptor-A antagonist, BQ-123, abolished the vasoconstriction effect of a short-term infusion of ET-1 in humans. The contribution of ET-1 to the evolution of the ischemic size in the heart was indirectly demonstrated by Watanabe et al,⁸ who showed that the use of specific antibodies neutralizing endogenous endothelin activity reduced the infarction size in rat hearts. In addition, Wang et al⁹ demonstrated in an anesthetized pig a local overproduction of endothelin-like immunoreactivity during myocardial ischemia/reperfusion, determining a potent coronary vasoconstriction. This effect was prevented by L-arginine infusion, with a mechanism possibly related to NO formation, which in turn was able to inhibit endothelin synthesis. Thus, a pharmacological approach aimed at decreasing endothelin levels might be a promising tool to reduce coronary vasoconstriction during acute myocardial ischemia in humans.

Intravenous heparin is a highly effective treatment for the control of myocardial ischemia in patients with unstable angina,^{10 11} even if correct monitoring of the coagulation factors is needed to avoid bleeding complications.¹² In recent years, the role of heparin in decreasing ET-1 levels has been demonstrated. In particular, Yokokawa et al¹³ found that long-term subcutaneous administration of heparin significantly decreased ET-1 levels in spontaneously hypertensive rats. Moreover, heparin suppressed ET-1 mRNA expression in cultured human endothelial cells¹⁴ and inhibited baseline and stimulated ET-1 levels in cultured rat mesangial cells¹⁵ with a mechanism related to an enhancement of endothelium-derived NO-mediated vasodilator response.^{14 15}

On the other hand, insulin has been applied in current clinical practice to ameliorate damage from ischemia during exercise¹⁶ and acute myocardial infarction,^{17 18} but the influence of exogenous insulin administration in the evolution of myocardial infarction and stimulation of ET-1 release has not been completely elucidated. Previous in vitro studies have shown that pharmacological insulin concentrations in the medium increased ET-1 release from cultured vascular smooth muscle cells derived from human microarterioles,¹⁹ enhancing ET-1 mRNA levels by 2.3-fold over baseline values in bovine aortic endothelial cells.²⁰ On the other hand, contradictory results were present when the activity of insulin in stimulating ET-1 release was studied in vivo. In fact, studies performed in healthy subjects showed that physiological insulin levels do not stimulate ET-1 release,^{21 22} whereas supraphysiological insulin levels are required to determine a significant increase in ET-1 release.²¹ Moreover, Ferri et al²³ and Wolpert et al²⁴ found a significant increase in ET-1 levels during euglycemic hyperinsulinemic clamp in patients with non–insulin-dependent diabetes and obese subjects.

Therefore, the aim of the study was to evaluate the effects of a continuous intravenous low-dose heparin infusion on arterialized ET-1 release in normal healthy humans. The combined effect of heparin and insulin infusion on ET-1 release also was evaluated in the same subjects by the euglycemic hyperinsulinemic clamp technique. To test whether heparin might indirectly modify ET-1 through modification of NO levels, nitrate concentrations were also measured before and at the end of each period.

	Methods

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Subjects
All subjects gave informed consent to participate in the study, and the protocols were approved by the local Ethics Committee.

Study Protocols
Eleven normal subjects (9 men, 2 women) with a mean (±SD) age of 28±1 years, a mean weight of 68.2±3.2 kg, and a mean body mass index of 22.7±0.7 kg/m² were studied. They underwent two tests in random order with at least a 15-day interval.

Fig 1 shows the study design. Intravenous heparin (Sodic Heparin, Eparina Vister, Park Davis SpA; prime, 250 IU in 1 mL saline; continuous, 600 IU in 6 mL saline per hour; test 1) or saline (prime, 1 mL saline; continuous, 6 mL saline per hour; test 2) infusions were maintained at fasting insulin levels (heparin/saline infusion period) for 120 minutes and during an insulin infusion of 25 mU·kg^-1·h^-1 diluted in 6 mL saline per hour (insulin period) for 120 minutes. Infusions were performed through a 20-gauge plastic cannula (Abbocath T, Abbot, Ireland LTD) inserted into a large antecubital vein of one arm. During the insulin period, blood glucose levels were maintained at baseline values by means of a variable 20% glucose infusion²⁵ according to blood glucose measurements obtained every 5 minutes. In particular, a 20-gauge plastic cannula was inserted into a dorsal vein of one hand in a patient in the retrograde position, and the hand was placed in a Plexiglas box and maintained at 55°C. It is necessary to arterialize venous blood by heating the hand because the arterial blood glucose should be monitored to accurately infuse glucose to maintain blood glucose at baseline levels. However, obtaining arterial samples is difficult and is associated with some risks related to arterial cannulation. Many investigators have therefore used the arterialized venous blood samples obtained from a heated hand to measure blood glucose levels during a euglycemic hyperinsulinemic clamp.^{21 26 27 28} Under these conditions, no changes in hematocrit were observed at the end compared with the pretest values in both tests, suggesting that the parameters under evaluation did not change by a simple dilution factor caused by liquid infusions.

View larger version (23K): [in this window] [in a new window]   Figure 1. Study design. inf indicates infusion; *, blood samples; and §, blood flow measurements.

ET-1 and NO samples were withdrawn simultaneously at -5, 0, 115, 120, 235, and 240 minutes. Samples for serum insulin determination were drawn at -5, 0, 30, 60, 90, 100, 110, 120, 150, 180, 210, 220, 230, and 240 minutes. Samples for the measurements of aPTT and PT were withdrawn immediately before and 120 and 240 minutes after the start of the test.

Blood flow of the proximal forearm was determined by use of venous occlusion plethysmography as reported elsewhere²⁹ immediately before the start of the study and at 105, 110, 115, 120, 225, 230, 235 and 240 minutes. All hemodynamic measurements were obtained with subjects in supine position. Blood flow was determined as milliliter per minute per 100 mL forearm volume tissue.

Assays
Blood glucose was measured with a glucose oxidase–based analyzer (Yellow Springs Instruments). Serum insulin levels (intra-assay coefficient of variation, 3.0%; interassay coefficient of variation, 5.0%) were measured by commercially available radioimmunoassay kits (Insulin I125 Ria kit, Incstar Corp). ET-1 samples were measured with a different commercial radioimmunoassay kit (Biomedica Gruppe) as reported previously.²¹ To evaluate whether heparin and/or insulin might interfere with the radioimmunoassay of ET-1, increasing doses of heparin (1, 5, and 10 U/mL) were added to the plasma samples in the absence or presence of increasing insulin levels (125, 250, 500, and 1000 µU/mL). In both conditions, measurement of the known concentrations of ET-1 in the samples (range, 3.76 to 33.4 pg/mL) was not influenced by the presence of increasing concentrations of heparin and insulin, with recovery ranging between 98% and 108%.

NO levels were evaluated through the measurement of the end products of their metabolism, ie, nitrite and nitrate levels, by use of enzymatic catalysis coupled with Griess reaction as reported previously.³⁰ No interferences in NO levels were found by the addition of increasing insulin and heparin levels in the range previously reported.

Statistical Analysis
Each variable was expressed as mean±SEM. Comparisons between experiments and among periods were made by use of ANOVA for a two-factor experiment. Pearson regression analyses were also performed. Values of P<.05 were considered statistically significant.

	Results

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During the heparin infusion period, blood glucose (4.8±0.1 versus 4.8±0.1 mmol/L) and serum insulin (78.2±16.8 versus 80.4±12.6 pmol/L) levels were similar in tests 1 and 2. Heparin infusion (test 1) decreased ET-1 levels by 32% (3.52±0.60 to 3.02±0.73 pg/mL), whereas NO and forearm blood flow increased by 29% (22.50±1.56 to 29.13±2.12 µmol/L) and 14% (3.12±0.10 to 3.55±0.14 mL/100 mL forearm tissue per minute), respectively (Fig 2). During the saline infusion period, ET-1, NO, and blood flow remained unchanged (Fig 2). There was a significant interaction between the effect of decreasing ET-1 levels and the heparin treatment (F, 4.06; df, 3.30; P<.01), between the effect of increasing NO levels and the heparin treatment (F, 15.3; df, 3.30; P<.001), and between the effect of increasing forearm blood flow and the heparin treatment (F, 4.11; df, 3.30; P<.01). The decrease in ET-1 levels significantly correlated with the increment in forearm blood flow in test 1 (r=.74; P<.01) but not in test 2 (Fig 3).

View larger version (25K): [in this window] [in a new window]   Figure 2. Arterial ET-1 levels (top), NO levels (middle), and forearm blood flow (bottom) during intravenous heparin (test 1) and saline (test 2) infusions.

View larger version (20K): [in this window] [in a new window]   Figure 3. Relationship between the decrease in ET-1 levels (x axis) and the increment in forearm blood flow (y axis) during heparin (test 1) and saline (test 2) infusions.

During the insulin infusion period, blood glucose was successfully clamped at the fasting levels with a coefficient of variation <3%, and insulin levels were 226.7±24.3 and 231.6±13.3 pmol/L in tests 1 and 2, respectively. Interestingly, simultaneous infusion of heparin and insulin (test 1) did not cause the expected additional decrease in ET-1 levels. On the contrary, ET-1 levels increased by 25%, returning to fasting levels. NO levels increased by 12%, and forearm blood flow remained unchanged (Fig 2). During test 2, insulin infusion caused a slight increase in ET-1 and NO levels, with no changes in forearm blood flow. No significant interactions were found between increasing ET-1 levels and insulin treatment. In contrast, there was a significant interaction between increasing NO levels and insulin treatment (F, 11.2; df, 3.30; P<.001).

The low-dose heparin infusion did not cause changes in the coagulation parameters like aPTT and PT (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study was designed to evaluate the effects of prolonged heparin infusion on ET-1 release in the presence of low insulin levels or mimicking postprandial insulin levels in normal subjects.

Our findings showed that a low-dose (600 IU/h) heparin infusion decreased arterial ET-1 levels and increased forearm blood flow in humans. The significant correlation between the decrease in ET-1 levels and the increase in forearm blood flow during heparin infusion (Fig 2) strongly suggests a causal link between the amelioration in vascular tone and a relatively small decrease in forearm arterial ET-1 levels. In fact, the effect of increased ET-1 levels in the induction of an exaggerated vasoconstriction is well known.^{31 32} The fact that heparin infusion significantly increased NO levels is the first in vivo evidence of the pathway followed by heparin in modulating vasodilatation. This finding is in agreement with previous in vitro studies in which heparin suppressed both baseline and thrombin-stimulated ET-1 mRNA expression and concomitantly enhanced NO formation and cGMP production in human endothelial cells.¹⁴ Moreover, significant decreases in ET-1, intracellular Ca²⁺, and inositol 1,4,5 triphosphate concentrations were demonstrated in cultured vascular smooth cells derived from aorta of spontaneously hypertensive rats after incubation with heparin ranging between 10 and 100 U/mL.¹³

In the present study, we did not test whether heparin, at the relative low doses used, modified thrombin production, but the fact that aPTT or PT activity remained unchanged suggests that coagulation cascade was not highly influenced. However, further studies are needed to properly evaluate the relationship of a dose-response curve of heparin action on the coagulation cascade and the decrease in ET-1 levels.

Interestingly, in the present study, an acute increase in insulin levels to mimic a postprandial period (200 pmol/L) reverted the inhibitory effect of heparin on ET-1 release, which returned to fasting concentrations with a concomitant increase in NO levels. Slight increases in ET-1 and NO levels also were demonstrated during insulin infusion in test 2. In our opinion, these data suggest that in the presence of mild hyperinsulinemia, both vasoconstrictor (ET-1) and vasodilator (NO) factors are stimulated, resulting in opposite effects on forearm blood flow, which in turn remains unchanged as previously demonstrated.³³ The effect of hyperinsulinemia on ET-1 release is an intriguing and stimulating result in keeping with previous data obtained by our group in normal subjects.²¹ In particular, in our previous study, an insulin bolus was performed that achieved a serum insulin peak of 4000 to 5000 pmol/L. Under those conditions, there was a significant increase in ET-1 levels, which nearly doubled 10 minutes after insulin administration. Moreover, in the same study, a slight increase in ET-1 levels was found at insulin levels of 200 pmol/L, but at insulin levels of only 1000 pmol/L, ET-1 levels significantly increased compared with baseline levels. Also under those conditions, no changes in forearm blood flow and vascular resistance were observed. To significantly decrease forearm blood flow, it was necessary to induce an acute and simultaneous increase in insulin (at 1000 pmol/L) and triglyceride (6 mmol/L) levels, which in turn increased ET-1 levels by 100%. The fact that in both the present and previous studies insulin infusion did not determine significant changes in forearm blood flow is only somewhat surprising because we know that it is necessary to acutely increase ET-1 baseline levels sevenfold to elicit an acute increase in blood pressure.³⁴ However, it is particularly interesting that a significant increase in arterial blood pressure was observed in patients with acute myocardial infarction³⁵ and angina pectoris³⁶ when glucose, insulin, and potassium were administered.

Some important clinical implications might be derived from our data. First, it is critical to reevaluate the effect of heparin in healthy human subjects because of its potential application in clinical practice. In fact, thrombolitic therapy is widely used in the treatment of myocardial infarction,³⁷ and the evidence that timely thrombogenesis significantly decreases short-term and long-term mortality is compelling. However, the use of thrombolytic agents at the usual dose of 5000 IU as intravenous bolus followed by a rate of 1000 IU/h entails some risk of bleeding,^{37 38 39 40} along with the desired pharmacological effect of activation of fibrinolytic system. The use of low-dose heparin infusion might be a promising tool to decrease the contribution of ET-1 in the induction of vasoconstriction during acute coronary events while avoiding the side effects of therapy. Moreover, it seems mandatory to take some precaution in the use of insulin in subjects affected by myocardial infarction because the real benefits were never proved and its effect on ET-1 release is a new and interesting finding. This fact appears even more important because it was reported previously that endothelin actively participates in the evolution of the infarction size of the ischemic heart.^{8 9 40} Furthermore, Omland et al⁴⁰ were able to demonstrate that plasma endothelin is an independent predictor of 1-year mortality after acute myocardial infarction.

In conclusion, the present study showed that low-dose heparin infusion decreases ET-1 levels with a significant increase in the forearm blood flow in humans. This effect seems mediated by a simultaneous increase in NO levels and is completely reversed by a mild increase in insulin concentrations. These are preliminary results, and further studies are necessary to evaluate the effects of prolonged insulin and heparin infusions on ET-1 and NO release before we can draw final conclusions.

	Selected Abbreviations and Acronyms

 

aPTT = activated partial thrombloplastin time ET-1 = endothelin-1 NO = nitric oxide PT = prothrombin time

Received July 15, 1996; revision received September 9, 1996; accepted September 24, 1996.

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