Citations

Introduction: ProANP is a 108 amino acid (AA) prohormone which is produced in the atria and processed to the mature 28 AA ANP which activates the guanylyl cyclase receptor-A (GC-A) and its second messenger cGMP. ProANP is found in the human circulation but its bioavailability is undefined. Hypothesis: ProANP represents a novel cardiac peptide with more sustained natriuretic actions than either carperitide (ANP) or nesiritide (BNP). ProANP itself can activate GC-A in vitro and the proANP can be processed in human serum from normals and in patients with heart failure (HF). Methods: We examined the in vivo cardiorenal actions of proANP compared to ANP, BNP, or placebo in normal canines (667 pmol/kg, n=5/group). We determined cGMP activation of proANP, ANP and BNP in GC-A expressing HEK293 cells. ProANP processing and degradation was also defined in serum from normal subjects (n=13) and patients with HF (n=14) ex vivo. Results: ProANP had significantly greater diuretic and natriuretic properties with increased renal blood flow and sustained renal tubular actions compared to ANP or BNP in canines. ProANP also resulted in greater and more prolonged cardiac unloading than ANP, and much less hypotensive effects than BNP. The plasma half-life of proANP was 4.5 times longer than ANP and 3.8 times longer than BNP. ProANP itself stimulated cGMP generation in GC-A expressing HEK cells which was equal to ANP. ProANP was processed to ANP in fresh serum from normals as well as HF patients ex vivo and the processed peptide activated cGMP in GC-A expressing HEK cells. Conclusions: ProANP represents a novel GC-A activator with a longer half-life and greater natriuresis and diuresis than ANP and BNP and less hypotensive effects than BNP. The bioactivity of exogenous proANP may be through direct stimulation of GC-A, as well as through processing into ANP, as proANP is processed in the circulation of both normal and HF. As proANP circulates in the human circulation, proANP may represent a key circulating hormone in cardiorenal and blood pressure homeostasis as well as a potential innovative therapeutic beyond ANP (carperitide) or BNP (nesiritide) for cardiorenal diseases including HF.

Decreased renal function is associated with increased CV morbidity and mortality. The changes mild renal impairment may cause structurally/molecularly in the heart remain unclear. We hypothesized that mild renal dysfunction produced by unilateral nephrectomy(UNX)results in changes in ventricular structure and gene expression in the absence of hypertension or vol overload. Wistar rats were used[sham (S; n=10) and UNX; n=9]. At 4 weeks, GFR was determined by inulin clearance and RBF by PAH. Hearts and kidneys were harvested for histological analysis. Cardiac function assessed by echo. Genomewide microarray analysis was performed on LV myocardium (Affymetrix). Glomerular hypertrophy was observed in UNX. GFR tended to decrease with a reduction in RBF (S:8±1, Nx:5±1 ml/min, p<0.005). Blood pressure (BP), Na and water excretions were maintained without activation of PRA, aldo or BNP. LVEF and LV diameters were normal. Microarray analysis of LV revealed that 278 genes changed with UNX (1.5 fold, P<0.05) related to cell growth, bone remodeling and muscle contraction (Z value>2). Importantly, Pic Red staining in the LV of UNX revealed greater fibrosis (S:2±0.1, Nx:4±0.4 %, p<0.001). We conclude that mild renal insufficiency initiates myocardial gene responses and fibrosis independent of alterations in the circulating RAAS, BNP, BP, vol overload or systolic dysfunction.

Studies in human myocardial infarction (MI) suggest that even in the absence of heart failure (HF) alterations in renal function may occur and contribute to poor outcomes. We hypothesized that even without preexisting renal disease, renal functional, structural and molecular changes would be present following MI. Wistar rats were used, Sham(S;n=10) and MI(n=9). 3 weeks after MI, GFR was determined by inulin clearance. Blood was obtained for PRA and aldosterone. Cardiac function was assessed by echo. Genomewide microarray analysis was performed on kidney cortex (KC) and medulla (KM) (Affymetrix). EF decreased after MI (S:63±2, MI:43±6%, p<0.01) and LVEDd increased. PRA, aldosterone and blood pressure (BP) did not change. There was no HF as Na and water excretion was maintained. GFR tended to decrease. Pic Red staining in the KC and KM after MI showed greater fibrosis (KC S:1±0.2, MI:3±0.6%, p<0.001 and KM S:1±0.2, MI:19±6%, p<0.005). Microarray analysis showed that 303 genes changed in KM and 407 genes in KC after MI (1.5 fold, P<0.05) related to structure morphogenesis and metabolic processes(Z value>2). We conclude that experimental MI results in renal structural remodeling characterized by cortical and medullary fibrosis with a mild reduction in GFR and extensive modulation of molecular pathways related to renal growth and morphogenesis underscoring a heart‐kidney connection after MI.

Background CBB‐NP is a novel fully humanized designer natriuretic peptide (NP) integrating the 17‐amino‐acid ring of human C‐type NP (CNP) with the N‐ and the C‐termini of human B‐type NP (BNP). CBB‐NP exerted natriuretic, diuretic, renal‐preserving and cardiac‐unloading actions in anesthetized dogs. In this investigation, we compared for the first time the cGMP stimulating actions of CBB‐NP vs human atrial NP (ANP), BNP and CNP in human aortic endothelial cells (HAEC). Methods CBB‐NP was synthesized (SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH with a disulfide bond joining the C residues). HAEC were incubated with ANP, BNP, CNP or CBB‐NP for 10 min (10 −6 M). Cyclic GMP was quantified by RIA. Results CBB‐NP and CNP increased cGMP (pmol/mL) vs control (0.108±0.020 and 0.174±0.036 vs 0.001±0.001, P<0.01 and <0.001, respectively). CBB‐NP elicited a greater cGMP response vs ANP (0.027±0.002, P<0.05), whereas comparable responses were observed vs BNP and CNP. When the native NPs were compared, a greater cGMP stimulating response was detected with CNP vs ANP and BNP (P<0.001 and <0.01, respectively). Conclusions The novel designer NP, CBB‐NP, stimulates cGMP in human aortic endothelial cells to a comparable extent as compared to the native BNP and CNP. Moreover, its cGMP stimulating action is greater than that of ANP. The therapeutic potential of CBB‐NP in models of cardiovascular diseases warrants further investigation. Supported by NIH, HFSA and ASCPT

Decreased renal function is associated with increased CV morbidity and mortality. However, the impact of mild renal dysfunction upon the heart remains to be fully elucidated. It is thought that a mild reduction in renal mass is not deleterious to the heart, yet exploration of the impact of unilateral nephrectomy (Nx) on myocardial gene responses is undefined. We hypothesized that even when ventricular function and structure are grossly preserved after Nx, such a reduction in renal mass and function would result in significant alterations in the cardiac genome. Wistar rats, Sham(S)(n=10), Nx(n=9). 4 weeks after Nx we assessed proteinuria, UNaV, UcGMP and BNP. Cardiac function was assessed by echo. GFR was determined by inulin clearance and renal blood flow (RBF) by PAH. Blood was obtained for BNP, aldosterone and cGMP. Kidneys and hearts were harvested. Gene microarrays were conducted of left ventricle (LV) myocardium (Affymetrix GeneChip® Rat Genome 230 2.0). GFR strongly tended to decrease and RBF was decreased in Nx (S:8±1, Nx:5±1 ml/min, p<0.005). Glomerular volume was increased in Nx (S:1±0.04, Nx:1.6±0.1 u 3 ×10 6 , p<0.001) while UNaV and volume were not different. There was no proteinuria in Nx. EF, LV end systolic and diastolic diameters were normal. Blood pressure (BP) was unchanged. There was no activation of the systemic RAAS or BNP. Importantly, 278 cardiac genes changed their expression after Nx (1.5 fold, P<0.05). 11% were increased and 89% decreased. Pathways involved included cell adhesion, growth and proliferation. We conclude that Nx, independently of alterations in circulating RAAS or BNP, BP or myocardial structure and function, results in widespread changes in myocardial gene expression. Further long‐term studies are needed to define the impact of these early gene responses to myocardial structure and function.

Mutant v-erbB products of avian c-erbB1 have previously been used to correlate structural domains of the receptor encoded by this proto-oncogene with tissue-specific transformation potential. In these studies, deletion of the ligand-binding domain of the receptor has been shown to be required for transformation of erythroblasts, fibroblasts, and endothelial cells. It has, therefore, been postulated that deletion of this domain results in an allosteric change in the receptor analogous to the ligand-bound state of the epidermal growth factor receptor; i.e., it induces a receptor conformation that is constitutively active with respect to mitogenic signaling. While oncogenic v-erbB products have been shown to be expressed on the cell surface of both fibroblasts and erythroblasts, no comprehensive analysis of the oligomeric potential of these products has been conducted. Since the first event known to follow epidermal growth factor binding to its receptor is oligomerization, and receptor dimerization has been correlated with mitogenic signaling, we have carefully analyzed the ability of several v-erbB products to oligomerize in the three target cell types transformed by these oncogenes. In this report, we demonstrate the v-erbB products can efficiently homodimerize in all three target tissues, that this dimerization is ligand independent and occurs at the cell surface, and that there is no apparent correlation between v-erbB dimerization and transformation of avian fibroblasts. Furthermore, both oncogenic and nononcogenic v-erbB products can heterodimerize with the native c-erbB1 product in chicken embryo fibroblasts, suggesting that heterodimerization between v-erB and native c-erbB1 is not sufficient to result in c-erbB1-mediated sarcomagenesis.

Abstract Introduction The rapid increase of patients of heart failure (HF) is a major health burden worldwide. Most importantly is the need to develop innovative new drugs for treatment of HF, such as sacubitril/valsartan which in part functions by enhancing the natriuretic peptides (NPs). We engineered NPA7 as a novel 30 amino acid bispecific designer peptide which activates the particulate guanylyl cyclase A receptor (pGC-A)/cGMP and for which the NPs both ANP and BNP are ligands and the Mas-receptor (MasR)/cAMP pathways for Angiotensin 1–7 (Ang1–7) is the endogenous ligand. We previously reported that acute intravenous (IV) administration of NPA7 shows cardiorenal protective and renin-aldosterone suppressing actions that go beyond the native peptides, BNP or Ang 1–7, which may have therapeutic potential for HF. Purpose To support the clinical development of NPA7 as a potential therapy in HF which promotes NP and MasR pathways, we investigated the actions and stability of subcutaneous (SQ) administration of NPA7 in normal canines. We also defined NPA7's peptide stability and metabolites in canine plasma. Methods Plasma and urinary cGMP, cardiorenal and renin-aldosterone responses to SQ injection (10μg/kg) were determined over 4 hours in normal canines (n=5) in vivo. Ex vivo, we established stability of NPA7 and key metabolites in canine serum using liquid chromatography-mass spectrometry (LC-MS). Data are expressed as mean ± SEM. * P<0.05 vs. BL. Results In vivo, SQ NPA7 resulted in a sustained increase at 2 hours in plasma (BL: 10±3; 120 min: 30±6* pmol/ml) and urinary (BL: 1033±198; 120 min: 5792±857* pmol/min) cGMP, GFR (BL: 29±6; 120 min: 70±12* ml/min) and sodium excretion (BL: 18±10; 120 min: 144±33* ueq/min). We observed a gradual reduction in BP at 60 min (BL: 109±4; 60 min: 99±7* mmHg) with a sustained decrease in PCWP at 4 hours (BL: 5±0.9; 240 min: 3.1±0.6* mmHg). SQ NPA7 also suppressed plasma renin and aldosterone up to 3 hours after SQ injection. LC-MS revealed that NPA7 was highly stable with both the pGC-A and MasR activating moieties intact ex vivo in canine serum with a disappearance time of 2 hours. We also identified 2 major NPA7 metabolites NPA71–27 and NPA71–28. Conclusions SQ NPA7 possesses cGMP activating, cardiac unloading, diuretic, natriuretic, and renin-aldosterone suppressing actions in normal canines. NPA7 is also highly stable in serum. These studies support SQ administration as an effective delivery strategy for NPA7, a first-in-class innovative bispecific dual pGC-A/MasR activator now in preclinical development for HF.

The natriuretic peptides (NPs) B-type NP (BNP) and urodilatin (URO) exert renal protective properties via the particulate guanylyl cyclase A receptor (pGC-A). As a potential renal-enhancing strategy, we engineered a novel designer peptide that we call CRRL269. CRRL269 was investigated in human cell lines and in normal canines to define potential cardiorenal enhancing actions. The mechanism of its cardiorenal selective properties was also investigated. In vitro NP receptor activity was quantified with guanosine 3′,5′-cyclic monophosphate generation. In vivo effects were determined in normal canine acute infusion studies. We observed that CRRL269 demonstrated enhanced pGC-A activity in renal compared with nonrenal cell lines. CRRL269 exerted enhanced resistance to neprilysin compared with URO. Importantly, CRRL269 exhibited significant and greater increases in urinary sodium excretion and diuresis, with less blood pressure reduction, than BNP or URO in normal canines. CRRL269 retained potent renin-angiotensin-aldosterone system (RAAS) suppressing properties shared by URO and BNP. Also, CRRL269 exerted less arterial relaxation and higher cAMP cardiomyocytes generation than BNP. CRRL269 possessed superior renal and pGC-A activating properties compared with BNP or URO in vitro. CRRL269 exerted enhanced renal actions while suppressing RAAS in vivo and with less hypotension compared with URO or BNP. Together, our study suggests that CRRL269 is a promising innovative renal-enhancing drug, with favorable protective actions targeting cardiorenal disease states through the pGC-A receptor.