Chen, Horng H.

Background—Neutral endopeptidase 24.11 (NEP) is a metalloprotease that is localized in the greatest abundance in the kidney and degrades natriuretic peptides, such as atrial natriuretic peptide (ANP). Mild congestive heart failure (CHF) is characterized by increases in circulating ANP without activation of the renin-angiotensin-aldosterone system (RAAS) or sodium retention. In contrast, severe CHF is characterized by sodium retention and coactivation of both ANP and the RAAS.Methods and Results—We defined the acute cardiorenal actions of the NEP inhibitor candoxatrilat (8 μg · kg−1· min−1) in 4 groups of anesthetized dogs (normal, n=8; mild CHF, n=6; severe CHF, n=5; and severe CHF with chronic AT1receptor antagonism, n=5). Mild CHF was produced by rapid ventricular pacing at 180 bpm for 10 days and severe CHF at 245 bpm for 10 days. In mild CHF, urinary sodium excretion and glomerular filtration rate were greatest in response to acute NEP inhibition compared with the response in either control animals or those with severe CHF. Furthermore, an increase in glomerular filtration rate was observed only in mild CHF in association with increases in renal blood flow and decreases in renal vascular resistance and distal tubular sodium reabsorption. Urinary ANP and cGMP excretion, markers for renal biological actions of ANP, were greatest in mild CHF. The renal actions observed in mild CHF were attenuated in severe CHF and not restored by chronic AT1receptor antagonism.Conclusions—The results of the present study demonstrate that acute NEP inhibition in mild CHF results in marked increases in renal hemodynamics and sodium excretion that exceed that observed in control animals and severe CHF. These studies underscore the potential therapeutic role for NEP inhibition to enhance renal function in mild CHF, an important phase of CHF that is marked by selective activation of endogenous ANP in the absence of an activated RAAS.

Mild heart failure is characterized by increases in atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in the absence of activation of the renin-angiotensin-aldosterone system (RAAS). Vasopeptidase (VP) inhibitors are novel molecules that coinhibit neutral endopeptidase 24.11, which degrades the natriuretic peptides (NPs) and ACE. In a well-characterized canine model of mild heart failure produced by ventricular pacing at 180 bpm for 10 days, we defined the renal and humoral actions of acute VP inhibition with omapatrilat (OMA, n=6) and acute ACE inhibition (n=5) alone with fosinoprilat. We also sought to determine whether the NPs participate in the renal actions of acute VP inhibition by the administration of OMA together with an intrarenal administration of the NP receptor antagonist HS-142-1 (n=5). OMA resulted in a greater natriuretic response than did ACE inhibition in association with increases in plasma cGMP, ANP, BNP, urinary cGMP, urinary ANP excretion, and glomerular filtration rate ( P <0.05 for OMA versus ACE inhibition). Plasma renin activity was increased only in the group subjected to ACE inhibition. Administration of intrarenal HS-142-1 attenuated the renal properties of OMA in association with a decrease in urinary cGMP excretion despite similar increases in plasma ANP and BNP. This study provides new insight into a unique new pharmacological agent that has beneficial renal actions in experimental mild heart failure beyond the actions that are observed with ACE inhibition alone and that are linked to the NP system.

Background— Preclinical diastolic dysfunction (PDD) has been broadly defined as subjects with left ventricular diastolic dysfunction, without the diagnosis of congestive heart failure (HF) and with normal systolic function. Our objective was to determine the risk factors associated with the progression from PDD (stage B) HF to symptomatic (stage C) HF. Methods and Results— Using the resources of the Rochester Epidemiology Project, all residents of Olmsted County, MN, who underwent echocardiography between January 1, 2004, and December 31, 2005, and had grade 2–4 diastolic dysfunction and ejection fraction ≥50% were identified. Patients with a diagnosis of HF before or within 30 days of the echocardiogram were excluded. Patients were also excluded if they had a diagnosis of atrial fibrillation or severe mitral or aortic valve regurgitation at the time of the echocardiogram. A total of 388 patients met the inclusion criteria. The mean age of the cohort was 67±12 years, with a female (57%) predominance. Prevalence of renal insufficiency (estimated glomerular filtration rate <60 mL/min per 1.73 m 2 ) was 34%. The 3-year cumulative probabilities of development of (stage C) HF, development of atrial fibrillation, cardiac hospitalization, and mortality were 11.6%, 14.5%, 17.7%, and 10.1% respectively. In multivariable Cox proportional hazard regression analysis, we determined that age, renal dysfunction, and right ventricular systolic pressure were independently associated with the development of HF. Conclusions— This population-based study demonstrated that in PDD (stage B) HF, there was a moderate degree of progression to symptomatic (stage C) HF over 3 years, and renal dysfunction was associated with this progression independent of age, sex, hypertension, coronary disease, and ejection fraction.

Impaired renal function with loss of nephron number in chronic renal disease (CKD) is associated with increased cardiovascular morbidity and mortality. However, the structural and functional cardiac response to early and mild reduction in renal mass is poorly defined. We hypothesized that mild renal impairment produced by unilateral nephrectomy (UNX) would result in early cardiac fibrosis and impaired diastolic function, which would progress to a more global left ventricular (LV) dysfunction. Cardiorenal function and structure were assessed in rats at 4 and 16 wk following UNX or sham operation (Sham); ( n = 10 per group). At 4 wk, blood pressure (BP), aldosterone, glomerular filtration rate (GFR), proteinuria, and plasma B-type natriuretic peptide (BNP) were not altered by UNX, representing a model of mild early CKD. However, UNX was associated with significantly greater LV myocardial fibrosis compared with Sham. Importantly, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed increased apoptosis in the LV myocardium. Further, diastolic dysfunction, assessed by strain echocardiography, but with preserved LVEF, was observed. Changes in genes related to the TGF-β and apoptosis pathways in the LV myocardium were also observed. At 16 wk post-UNX, we observed persistent LV fibrosis and impairment in LV diastolic function. In addition, LV mass significantly increased, as did LVEDd, while there was a reduction in LVEF. Aldosterone, BNP, and proteinuria were increased, while GFR was decreased. The myocardial, structural, and functional alterations were associated with persistent changes in the TGF-β pathway and even more widespread changes in the LV apoptotic pathway. These studies demonstrate that mild renal insufficiency in the rat results in early cardiac fibrosis and impaired diastolic function, which progresses to more global LV remodeling and dysfunction. Thus, these studies importantly advance the concept of a kidney-heart connection in the control of myocardial structure and function.

Myocardial aging is characterized by left ventricular (LV) fibrosis leading to diastolic and systolic dysfunction. Studies have established the potent antifibrotic and antiproliferative properties of C-type natriuretic peptide (CNP); however, the relationship between circulating CNP, LV fibrosis, and associated changes in LV function with natural aging are undefined. Accordingly, we characterized the relationship of plasma CNP with LV fibrosis and function in 2-, 11-, and 20-month–old male Fischer rats. Further in vitro, we established the antiproliferative actions of CNP and the participation of the clearance receptor using adult human cardiac fibroblasts. Here we establish for the first time that a progressive decline in circulating CNP characterizes natural aging and is strongly associated with a reciprocal increase in LV fibrosis that precedes impairment of diastolic and systolic function. Additionally, we demonstrate in cultured adult human cardiac fibroblasts that the direct antiproliferative actions of high-dose CNP may involve a non-cGMP pathway via the clearance receptor. Together, these studies provide new insights into myocardial aging and the relationship to the antifibrotic and antiproliferative peptide CNP.

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.

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.

Background— Renal insufficiency is associated with increased morbidity and mortality after cardiopulmonary bypass cardiac surgery. B-type natriuretic peptide is a cardiac hormone that enhances glomerular filtration rate and inhibits aldosterone. Cystatin has been shown to be a better endogenous marker of renal function than creatinine. Methods and Results— We performed a double-blinded placebo-controlled proof of concept pilot study in patients (n=40) with renal insufficiency preoperatively (defined as an estimated creatinine clearance of <60 mL/min determined by the Cockroft-Gault formula), undergoing cardiopulmonary bypass cardiac surgery. Patients were randomized to placebo (n=20) or IV low dose nesiritide (n=20; 0.005 μg/Kg/min) for 24 hours started after the induction of anesthesia and before cardiopulmonary bypass. Patients in the nesiritide group had an increase of plasma B-type natriuretic peptide and its second messenger cGMP with a decrease in plasma cystatin levels at the end of the 24-hour infusion. These changes were not observed in the placebo group. There was a significant activation of aldosterone in the placebo group at the end of the 24-hour infusion, but not in the nesiritide group. At 48 and 72 hours, there was a decrease in estimated creatinine clearance and an increase in plasma cystatin as compared with end of the 24-hour infusion in the placebo group. In contrast, renal function was preserved in the nesiritide group with no significant change in estimated creatinine clearance and a trend for plasma cystatin to increase as compared with end of the 24-hour infusion. Conclusion— This proof of concept pilot study supports the conclusion that perioperative administration of low dose nesiritide is biologically active and decreases plasma cystatin in patients with renal insufficiency undergoing cardiopulmonary bypass cardiac surgery. Further studies are warranted to determine whether these physiological observations can be translated into improved clinical outcomes.