Effects of B-Type Natriuretic Peptide (Nesiritide) on Coronary
Epicardial Arteries, Systemic Vasculature and Microvessels

Nesiritide (Natrecor^®, Scios Inc., Fremont, California) is a recombinant form of human B-type (brain) natriuretic peptide (BNP) that has beneficial vasodilatory, diuretic and neurohormonal effects.¹ It activates membrane-bound guanylyl cyclase A-receptor, resulting in accumulation of intracellular cGMP, which in turn mediates the vasodilatory effects in vascular smooth muscle cells.²³ Nesiritide is manufactured from E. coli using recombinant DNA technology with a molecular weight of 3,464 g/mol with the same 32 amino acid sequence as the endogenous peptide. The mean terminal elimination half-life (t 1/2 ) of nesiritide is approximately 18 minutes and is cleared via binding to cell surface clearance receptors with subsequent cellular internalization and lysosomal proteolysis, proteolytic cleavage and renal filtration.

The drug is commonly used in the management of patients with decompensated congestive heart failure (CHF), since it has vasodilatory and natriuretic properties.^2,4,24 It has been shown to decrease pulmonary wedge pressures, as well as improve global clinical status.¹ Initial studies showed that nesiritide had a good safety profile, the primary side effects being headache and dose-related hypotension.^2,3
Recent reports, however, not only raise an issue of increased incidence of renal failure,²¹ but also up to a 50% increase in mortality.²² Given that the majority of patients with heart failure in the United States have ischemic heart disease, it is particularly important to further examine the effect of nesiritide on coronary vasculature. Endogenous BNP levels are increased in heart failure, but also in myocardial ischemia.4 It would be particularly important to exclude the effect of nesiritide on coronary vasculature as the cause of increased mortality due to phenomena such as “coronary steal” observed with nitroprusside.
The effect of intravenous nesiritide has been examined in normal human coronary arteries (5 patients) compared to diseased coronary arteries (5 patients with at least 1 coronary vessel with > 75% stenosis).¹¹ A decrease in coronary resistance and an increase in coronary diameter in patients with coronary disease in that small study was lower than that observed in normal controls, but the result was not statistically significant. Myocardial oxygen uptake was not significantly different in the two patient populations. Prior studies of the effect of nesiritide in pigs examined only the effect of intracoronary nesiritide.⁹ The purpose of this study was to evaluate the in vivo and in vitro effects of intravenous and intracoronary nesiritide on coronary circulation.

Materials and Methods
Animal preparation. Fourteen Yorkshire pigs of either sex (40.33 ± 4.18 kg in weight) were anesthetized with intramuscular ketamine hydrochloride (10 mg/kg) and isoflurane inhalation. Eight animals were used for hemodynamic monitoring and coronary measurements, and the additional 6 animals were used for microvascular analysis.The investigation was carried out according to a protocol approved by the Institutional Animal Committee, Beth Israel Deaconess Medical Center/Harvard Medical School. The treatment of animals was in accordance with the National Institutes of Health guidelines.
In vivo experiments. Eight Yorkshire pigs were used for this series of experiments. A right femoral cutdown was performed and an 8 Fr sheath was inserted. An 8 Fr Hockey Stick guiding catheter (HS-1, Cordis Corp., Miami Lakes, Florida) was used to engage the left main coronary artery. Under angiographic guidance, a 0.014 inch Doppler Flow Wire (Cardiometrics Inc., Mountain View, California) was advanced to the left anterior descending artery (LAD). A 2.9 Fr 40 MHz intravascular ultrasound catheter (CVIS, Scimed Life Systems, Maple Grove, Minnesota) was delivered over the Doppler Flow Wire and positioned in the mid LAD. This allowed for simultaneous measurement of the LAD cross-sectional area (CSA) and the average spectral peak velocity (APV) after: 1) intravenous administration of nesiritide (clinical dose: bolus of 2 μg/kg followed by a continuous infusion of 0.01 μg/kg/minute for 30 minutes); and 2) intracoronary nesiritide (ascending doses of 50, 100 and 200 μg). The hemodynamic parameters were allowed to return to baseline between each intervention. Systemic pressure and heart rate(HR) were measured and recorded throughout the experiment. APV and CSA were recorded simultaneously at baseline, 1, 5, 10, 15, 20 and 30 minutes after drug delivery.
Microvessel study. Microvascular reactivity experiments were conducted as previously described in 6 normal pigs.⁸ In brief, myocardial microvessels (arterioles: 60–100 μm in internal diameter) were dissected from the left ventricular myocardium and the right atrial wall. Microvessels were placed in an isolated plexiglass chamber cannulated with dual glass micropipettes measuring 30–80 μm in diameter and secured with 10-0 nylon monofilament suture (Ethicon, Somerville, New Jersey). Oxygenated (95% O₂, 5% CO₂) Krebs buffer solution warmed to 37ºC was continuously circulated through the organ chamber using a reservoir containing 100 ml. The vessels were pressurized to 40 mmHg in a no-flow state using a burette manometer filled with Krebs buffer solution. Using an inverted microscope (40–200 x, Olympus, Japan) connected to a video camera, the vessel image was projected onto a video monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, Vermont) was used to measure internal lumen diameter. Measurements were recorded with a stripchart recorder. Vessels were allowed to equilibrate for 30 minutes in Krebs buffer solution before an intervention and for 15 minutes between applications of each drug. Microvessels were precontracted by 30–60% of baseline with the thromboxane analogue U46619 (10^-9–10^-6 M). Microvascular responses to nesiritide and adenosine diphosphate (ADP, an endotheliumdependent vasodilator) were measured, both before and after pretreatment with 3 μM N^G-nitro-L-arginine.
Statistical analysis. Data are expressed as mean ± standard deviation. Continuous variables were compared by analysis of variance (ANOVA). Vascular responses were examined by ANOVA with the post hoc Fisher’s test. All reported p-values were two-tailed, and a p-value £ 0.05 was considered statistically significant.

Results
In vivo studies. In this portion of the study, changes in systemic blood pressure (SBP), heart rate (HR), coronary flow velocity (average spectral peak velocity, APV) and LAD crosssectional area (CSA) were recorded in response to intravenous (IV) and intracoronary (IC) nesiritide. The measurements were repeated after pretreatment with L-NAME to determine dependence on the nitric oxide (NO) pathway.

After IV nesiritide, HR increased at 1 minute and increased maximally at 30 minutes from 101.6 ± 8 to 109.6 ± 12. None of the increases in heart rate measured at 1-, 5-, 10-, 15-, 20- and 30-minute intervals were statistically different (p = 0.9711) (Figure 1A). SBP decreased maximally at 10 minutes from 89 ± 17 mmHg to 74 ± 9 mmHg (p = 0.6418). DBP decreased from 64.3 ± 17 mmHg to 56.3 ± 6.4 mmHg (p = 0.8189) at 10 minutes and then returned to baseline at 30 minutes. IV nesiritide did not have a significant (beneficial or adverse) effect on coronary flow velocity or vasodilatation at any measured time point (Figures 1 D, E and 2 A and B). APV decreased from 11.6 ± 1.15 to 10.8 ± 2.03 (p = 0.9995) at 10 minutes. CSA increased from 8.86 ± 2.3 to 9.2 ± 2.26 (p = 0.9999) at 30 minutes.
Intracoronary nesiritide resulted in a slight but not statistically significant decrease in SBP (9 mmHg at 10 minutes; p = 0.3113) from baseline. This was not affected by pretreatment with L-NAME. AVP increased (trend) only in response to a 100 μg bolus of nesiritide from 15.8 ± 7.8 to 19 ± 7.5 cm/second at 20 minutes after infusion (p = 0.6419). There was no effect of IC nesiritide on CSA at any time point or at any dose (Figure 3).

In vitro study. Coronary microvessels from the left ventricle and right atrium were used to assess the microvascular response to nesiritide and ADP (an endothelium-dependent vasodilator) before and after pretreatment with L-NAME (Figure 4). With a high concentration of 10^-5-4 M, nesiritide caused relaxation in left ventricular and right atrial microvessels. In the right atrium, the absolute diameter increased from 42.5–55.8, which corresponded to relaxation of 23.9 ± 17.1% (p = 0.009). This effect was less potent than ADP. The microvascular effect of nesiritide and ADP were partially inhibited by L-NAME. In the left ventricle, the maximal absolute vessel diameter changed from 40.5–49.25 in response to nesiritide, which corresponded to a 23 ± 12.88% relaxation (p = 0.079). This response may indicate that the vasodilatory effect of nesiritide on the endothelium was partially mediated by a NO-dependent process. However, nesiritide affects the endothelium also by a NOindependent pathway.

Discussion
We demonstrate that IV nesiritide resulted in a very minimal vasodilatory systemic response without adversely affecting coronary blood flow. The absence of reduction in coronary blood flow with the drop in systemic pressure suggests a favorable response in coronary circulation. IC administration of nesiritide had minimal effects on coronary flow and epicardial vessels. In vitro, nesiritide had a small vasodilatory effect on microvessels. The magnitudes of coronary epicardial vasodilation and augmentation in coronary blood flow in response to nesiritide were consistent with prior studies of IC infusion in pigs⁹ and humans.^10,11
BNP is thought to act via the second messenger cGMP on vascular smooth muscle cells. The GC-A receptor, a biological receptor for BNP, is part of a receptor class of proteins termed particulate guanylyl cyclase.¹⁰ It is a membrane-bound protein with guanylyl cyclase activity. Binding of BNP to the extracellular domain of the GC-A receptor activates the intracellular guanylyl cyclase domain, resulting in the catalysis of cGMP from GTP.¹⁰
Recent work indicates that natriuretic peptide signaling can attenuate the development of irreversible ischemic injury.¹² BNP-32 administration to isolated perfused rat hearts prior to and during left main coronary artery occlusion resulted in limitation of infarct size in a concentrationdependent fashion. This action of BNP was associated with an increase in myocardial cGMP concentration. An unexpected observation in that model was that the infarct-limiting action of BNP was attenuated by treatment with L-NAME, an inhibitor of NO synthase. This would suggest that activation of the NO-soluble guanylyl cyclase pathway is essential for the cytoprotective action of BNP.¹³ We also showed that the microvascular effect of nesiritide and ADP were partially inhibited by L-NAME, indicating that both effects were at least partially mediated by a NOdependent process. It is speculated7 that in disease characterized by increased production of natriuretic peptides, increased myocardial concentrations of cGMP are likely to be a pivotal second messenger in natriuretic peptide cytoprotective signaling and may be an important salvage mechanism in the event of ischemia.
Other natriuretic peptides may exert anti-ischemic actions in experimental models. Human recombinant ANP (carperitide) was found to limit infarct size in the dog in vivo.¹⁴ Urodilatin, a renal-derived natriuretic peptide, has been shown to limit infarct size in the pig heart in vivo.¹⁵ Thus, natriuretic peptide signaling may be an important injury limiting mechanism in the acutely ischemic myocardium.⁷
It has been shown that ANP and BNP modulate cell growth, apoptosis and proliferation in cardiac myocytes16 and smooth muscle cells,¹⁷ and suppress cardiac fibroblast proliferation¹⁸ and extracellular matrix secretion.^19,20 These potential anti-ischemic properties of BNP make them attractive contenders for further investigation as adjunctive treatments during acute coronary syndromes.⁷
In our study, we showed that a clinical dose of BNP does not adversely affect coronary hemodynamics, and its effect on systemic hemodynamics is transient. Therefore, patients with ischemic myocardium may benefit from treatments that promote microvascular vasodilatation and slow the rate of irreversible cellular injury during prolonged ischemic episodes without effects on epicardial vessels.⁷ Similar experiments with physiological intracoronary BNP and microvessel preparations should be conducted in models of coronary ischemia to further explore the effect of BNP on diseased coronary arteries.

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