Boerrigter, Guido

M-atrial natriuretic peptide (ANP; M-ANP) is a novel next generation 40 amino acid peptide based on ANP, which is highly resistant to enzymatic degradation and has greater and more sustained beneficial actions compared with ANP. The current study was designed to advance our understanding of the therapeutic potential of M-ANP in a canine model of acute angiotensin II–induced hypertension with elevated cardiac filling pressures and aldosterone activation. We compare M-ANP with vehicle and equimolar human B-type natriuretic peptide, which possesses the most potent in vivo actions of the native natriuretic peptides. M-ANP significantly lowered mean arterial pressure and systemic vascular resistance. Importantly, despite a reduction in blood pressure, renal function was enhanced with significant increases in renal blood flow, glomerular filtration rate, diuresis, and natriuresis after M-ANP infusion. Although angiotensin II induced an acute increase in pulmonary capillary wedge pressure, M-ANP significantly lowered pulmonary capillary wedge pressure, pulmonary artery pressure, and right atrial pressure. Further, M-ANP significantly suppressed angiotensin II–induced activation of aldosterone. These cardiovascular and renal enhancing actions of M-ANP were accompanied by significant increases in plasma and urinary cGMP, the second messenger molecule of the natriuretic peptide system. When compared with human B-type natriuretic peptide, M-ANP had comparable cardiovascular actions but resulted in a greater natriuretic effect. These results suggest that M-ANP, which is more potent than ANP in normal canines, has potent blood pressure lowering and renal enhancing properties and may, therefore, serve as an ANP based therapeutic for acute hypertension.

Background— Reductions in numbers of circulating progenitor cells (CD34+ cell subsets) have been demonstrated in patients at risk for, or in the presence of, cardiovascular disease. The mediators of these reductions remain undefined. To determine whether neurohumoral factors might regulate circulating CD34+ cell subsets in vivo, we studied complementary canine models of left ventricular (LV) dysfunction. Methods and Results— A pacing model of severe LV dysfunction and a hypertensive renal wrap model in which dogs were randomized to receive deoxycorticosterone acetate (DOCA) were studied. Circulating CD34+ cell subsets including hematopoietic precursor cells (HPCs: CD34+/CD45 dim /VEGFR2−) and endothelial progenitor cells (EPCs: CD34+/CD45−/VEGFR2+) were quantified. Additionally, the effect of mineralocorticoid excess on circulating progenitor cells in normal dogs was studied. The majority of circulating CD34+ cells expressed CD45dimly and did not express VEGFR2, consistent with an HPC phenotype. HPCs were decreased in response to pacing, and this decrease correlated with plasma aldosterone levels (Spearman rank correlation=−0.67, P =0.03). In the hypertensive renal wrap model, administration of DOCA resulted in decreased HPCs. No changes were seen in EPCs in either model. Normal dogs treated with DOCA exhibited a decrease in HPCs in peripheral blood but not bone marrow associated with decreased telomerase activity. Conclusions— This is the first study to demonstrate that mineralocorticoid excess, either endogenous or exogenous, results in reduction in HPCs. These data suggest that mineralocorticoids may induce accelerated senescence of progenitor cells, leading to their reduced survival and decline in numbers.

Background— Hemodynamic and neurohumoral function can affect the efficacy of diuretic therapy in congestive heart failure. Arginine vasopressin increases water reabsorption through the V 2 receptor in the collecting duct, whereas B-type natriuretic peptide (BNP) decreases sodium reabsorption in the collecting duct. We hypothesized that combining BNP to the V 2 -receptor antagonist tolvaptan (TLV) would enhance renal excretory function by augmenting sodium excretion together with aquaresis without adversely affecting renal hemodynamics in experimental congestive heart failure. Methods and Results— Congestive heart failure was induced in 3 groups (n=6 per group) of dogs by tachypacing. A acute experiment was done after 10 days. After baseline measurements, study groups received a 0.1 mg/kg IV bolus of TLV alone (TLV), TLV in combination with BNP (TLV+BNP; 50 ng/[kg · min]), or BNP alone (BNP). Mean arterial pressure increased with TLV, remained unchanged with TLV+BNP, and decreased with BNP (+5±1mm Hg versus −1±1 mm Hg versus −15±1 mm Hg; P <0.05). Renal blood flow and glomerular filtration rate were preserved with all regimens. Urine flow increased in all 3 groups but significantly more so with TLV+BNP (TLV: +0.4±0.1 mL/min versus TLV+BNP: +2.4±0.5 mL/min versus BNP: +0.8±0.3 mL/min; P <0.05). Only TLV+BNP and BNP were natriuretic ( P <0.05), whereas only TLV and TLV+BNP increased electrolyte-free water excretion ( P <0.05). Compared with TLV alone, TLV+BNP prevented an increase in aldosterone ( P <0.05). Conclusions— Coadministration of TLV and BNP in experimental HF resulted in a beneficial profile of renal, neurohumoral, and hemodynamic actions, specifically potent diuresis with natriuresis, neutral effect on mean arterial pressure, and lack of aldosterone activation.

Alternative RNA splicing may provide unique opportunities to identify drug targets and therapeutics. We identified an alternative spliced transcript for B-type natriuretic peptide (BNP) resulting from intronic retention. This transcript is present in failing human hearts and is reduced following mechanical unloading. The intron-retained transcript would generate a unique 34 amino acid (aa) carboxyl terminus while maintaining the remaining structure of native BNP. We generated antisera to this carboxyl terminus and identified immunoreactivity in failing human heart tissue. The alternatively spliced peptide (ASBNP) was synthesized and unlike BNP, failed to stimulate cGMP in vascular cells or vasorelax preconstricted arterial rings. This suggests that ASBNP may lack the dose-limiting effects of recombinant BNP. Given structural considerations, a carboxyl-terminal truncated form of ASBNP was generated (ASBNP.1) and was determined to retain the ability of BNP to stimulate cGMP in canine glomerular isolates and cultured human mesangial cells but lacked similar effects in vascular cells. In a canine-pacing model of heart failure, systemic infusion of ASBNP.1 did not alter mean arterial pressure but increased the glomerular filtration rate (GFR), suppressed plasma renin and angiotensin, while inducing natriuresis and diuresis. Consistent with its distinct in vivo effects, the activity of ASBNP.1 may not be explained through binding and activation of NPR-A or NPR-B. Thus, the biodesigner peptide ASBNP.1 enhances GFR associated with heart failure while lacking the vasoactive properties of BNP. These findings demonstrate that peptides with unique properties may be designed based on products of alternatively splicing.

Background— We previously reported the feasibility of an acute, orally delivered, newly developed, conjugated form of human B-type natriuretic peptide (hBNP) in normal animals. The objective of the present study was to extend our findings and to define the chronic actions of an advanced oral conjugated hBNP (hBNP-054) administered for 6 days on sodium excretion and blood pressure. We also sought to establish the ability of this new conjugate to acutely activate cGMP and to reduce blood pressure in an experimental model of angiotensin II (ANG II) –mediated hypertension. Methods and Results— First, we developed additional novel conjugated forms of oral hBNP that were superior to our previously reported hBNP-021 in reducing blood pressure in 6 normal dogs. We then tested the new conjugate, hBNP-054, chronically in 2 normal dogs to assess its biological actions as a blood pressure–lowering agent and as a natriuretic factor. Second, we investigated the effects of acute oral hBNP-054 or vehicle in 6 dogs that received continuous infusion of ANG II to induce hypertension. After baseline determination of mean blood pressure (MAP) and blood collection for plasma hBNP and cGMP, all dogs received continuous ANG II infusion (20 ng · kg −1 · min −1 , 1 mL/min) for 4 hours. After 30 minutes of ANG II, dogs received oral hBNP-054 (400 μg/kg) or vehicle in a random crossover fashion with a 1-week interval between dosing. Blood sampling and MAP measurements were repeated 30 minutes after ANG II administration and 10, 30, 60, 120, 180, and 240 minutes after oral administration of hBNP-054 or vehicle. In the chronic study in normal dogs, oral hBNP-054 effectively reduced MAP for 6 days and induced a significant increase in 24-hour sodium excretion. hBNP was not present in the plasma at baseline in any dogs, and it was not detected at any time in the vehicle group. However, hBNP was detected throughout the duration of the study after oral hBNP-054, with a peak concentration at 30 minutes of 1060±818 pg/mL. In the acute study, after ANG II administration, plasma cGMP was not activated after vehicle, whereas it was significantly increased after oral hBNP-054 ( P =0.01 between the 2 groups). Importantly, MAP was significantly increased after ANG II throughout the acute study protocol. However, although no changes occurred in MAP after vehicle administration, oral hBNP-054 reduced MAP for >2 hours (from 138±1 mm Hg after ANG II to 124±2 mm Hg at 30 minutes, 124±2 mm Hg at 1 hour, and 130±5 mm Hg at 2 hours after oral hBNP-054; P <0.001). Conclusions— This study reports for the first time that a novel conjugated oral hBNP possesses blood pressure–lowering and natriuretic actions over a 6-day period in normal dogs. Furthermore, hBNP-054 activates cGMP and reduces MAP in a model of acute hypertension. These findings advance the concept that orally administered chronic BNP is a potential therapeutic strategy for cardiovascular diseases such as hypertension.

Unlike healthy subjects, overt congestive heart failure cannot “escape” the sodium- and water-retaining actions of mineralocorticoid excess. It is undefined whether escape occurs in asymptomatic left ventricular dysfunction (ALVD), which is characterized by preserved sodium homeostasis, natriuretic peptide activation, and normal circulating aldosterone. We hypothesized that, in ALVD, mineralocorticoid excess with exogenous deoxycorticosterone acetate (DOCA) would overwhelm renal compensatory mechanisms, resulting in sodium and water retention, and promote renal and cardiac collagen deposition. ALVD was induced in 2 groups (N=5 each) of dogs by tachypacing at 180 bpm. Urine was collected daily and blood drawn at baseline and days 2, 5, 8, and 11. One group served as control (ALVD), and the other received DOCA (ALVD+DOCA) starting at day 2 of pacing. Urine flow and sodium excretion were unchanged in the ALVD group. In ALVD+DOCA, urine flow and sodium excretion decreased on the first 2 days DOCA was given but normalized starting day 4. Urine flow and urinary cGMP excretion increased in ALVD+DOCA after DOCA escape. Plasma atrial natriuretic peptide, B-type natriuretic peptide, and cGMP increased equally in both groups. There were no differences in cardiorenal and hemodynamic parameters in an acute study on day 11. Although renal collagen area fraction was similar, left ventricular collagen area fraction in ALVD+DOCA was significantly higher than in ALVD (3.3±0.4% versus 2.0±0.2%; P =0.012). We conclude that ALVD can escape the sodium- and water-retaining effects of mineralocorticoid excess. Despite renal escape, increased left ventricular collagen deposition suggests that the heart but not the kidney failed to escape the tissue effects of DOCA.

Recent studies support the speculation that different molecular forms of the cardiac hormone BNP with differential biological activity may circulate in heart failure and be detected by conventional assays. In the current study we determined the ability of 3 widely used conventional assays to detect these different forms thought to circulate in heart failure. We also evaluated the ability of pro-BNP (1-108), N-terminal peptide (NT)–pro-BNP (1-76), and BNP 3-32, the latter a cleavage product of BNP 1-32 by dipeptidyl peptidase IV, on an equimolar basis to activate cGMP in cultured cardiac fibroblasts and cardiomyocytes compared with the biologically active mature BNP 1-32. Specifically, we observed that the Roche NT–pro-BNP assay detected both NT–pro-BNP 1-76 and pro-BNP 1-108 and that Biosite Triage and Shionogi detected both mature BNP 1-32 and the shortened BNP 3-32. Moreover, in cultured cardiac fibroblasts and cardiomyocytes, BNP 1-32 (10 −6 mol/L) activated cGMP. BNP 3-32 demonstrated a similar cGMP activating property in both cardiac cell types. In contrast, the cGMP response to pro-BNP 1-108 and NT–pro-BNP 1-76 was not significantly greater than no treatment alone. We conclude that widely used commercial assays for NT–pro-BNP 1-76 and BNP 1-32 cannot differentiate among pro-, processed, or degraded forms and, thus, may not thoroughly identify circulating BNP forms in heart failure patients. These findings also demonstrate differential cGMP activating properties of BNP forms and, importantly, that pro-BNP 1-108 and NT–pro-BNP 1-76 have reduced cGMP activity in vitro that may have biological relevance to human heart failure.

Soluble guanylate cyclase is a heterodimeric enzyme with a prosthetic heme group that, on binding of its main ligand, NO, generates the second messenger cGMP. Unlike conventional nitrovasodilators, the novel direct NO- and heme-independent soluble guanylate cyclase activator BAY 58-2667 is devoid of non-cGMP actions, lacks tolerance development, and preferentially activates NO-insensitive heme-free or oxidized soluble guanylate cyclase. BAY 58-2667, therefore, represents a novel therapeutic advance in mediating vasodilation. To date, its cardiorenal actions in congestive heart failure (CHF) are undefined. We, therefore, hypothesized that BAY 58-2667 would have beneficial preload- and afterload-reducing actions in experimental severe CHF together with renal vasodilating properties. We assessed the cardiorenal actions of intravenous administration of 2 doses of BAY 58-2667 (0.1 and 0.3 μg/kg per minute, respectively) in a model of tachypacing-induced severe CHF. In CHF, BAY 58-2667 dose-dependently reduced mean arterial, right atrial, pulmonary artery, and pulmonary capillary wedge pressure (from baseline 19±1 to 12±2 mm Hg). Cardiac output (2.4±0.3 to 3.2±0.4 L/min) and renal blood flow increased. Glomerular filtration rate and sodium and water excretion were maintained. Consistent with cardiac unloading, atrial and B-type natriuretic peptide decreased. Plasma renin activity ( P =0.31) and aldosterone remained unchanged ( P =0.19). In summary, BAY 58-2667 in experimental CHF potently unloaded the heart, increased cardiac output and renal blood flow, and preserved glomerular filtration rate and sodium and water excretion without further neurohumoral activation. These beneficial properties make direct soluble guanylate cyclase stimulation with BAY 58-2667 a promising new therapeutic strategy for cardiovascular diseases, such as heart failure.