Sangaralingham, Jeson

Introduction: Therapeutic strategies dual-targeting renin-angiotensin system (RAS) and natriuretic peptide system (NPS), such as Sacubitril/Valsartan, have been proven promising in treating cardiorenal disease (CRD). However, mechanisms behind the crosstalk between RAS and NPS remain to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the receptor activated by ANP and BNP to increase its second messenger cGMP, has not been investigated. Hypothesis: We hypothesize ANGII naturally suppresses pGC-A activity and inhibition of this ANGII mediated action can be a new strategy to combat CRD. Methods: Studies consist of biomarker analyses in plasma from healthy humans, drug infusions in normal rats, molecular characterization in human HEK293 cells, and in vitro assessment of CRRL290, an innovative dual-functional peptide designed to activate pGC-A and inhibit protein kinase C (PKC). Results: In 128 healthy humans, plasma cGMP was positively associated with ANP or BNP in low (≤ 4.5 pg/mL) but not those with high (> 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII. Interestingly, suppression effect of ANGII on pGC-A was only recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. We also found PKC, a known downstream target of AT1R, was a key mediator as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Accordingly, we designed and synthesized CRRL290. Notably, CRRL290 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in HEK293 cells. Conclusions: We demonstrate that ANGII is a natural suppressor of pGC-A via AT1R and PKC signaling. We also report for the first time that CRRL290, a novel bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Our study provides new insights into the RAS-NPS interaction, and highlights a novel and mechanism-based therapeutic avenue for treating CRD.

Introduction: The cardiac hormone atrial natriuretic peptide (ANP) plays a key role in blood pressure (BP) and metabolic homeostasis through guanylyl cyclase receptor-A. While the role of ANP in cardiovascular disease (CVD) has been extensively investigated, its role in metabolic regulation has only recently emerged. A genetic variant of the ANP gene (rs5068), which increases production of ANP and NT-proANP, is associated with lower blood pressure (BP), reduced risk for hypertension and a decreased prevalence of obesity and metabolic syndrome. Hypothesis: We hypothesized that sex differences in ANP may have an important role in cardiometabolic disease together with cardiovascular (CV) morbidity and mortality. Methods: We studied 1176 subjects (age > 45 years) in the general population from Olmsted County MN. Plasma ANP molecular forms were quantified. Metabolic and cardiorenal parameters, and detailed medical records were reviewed. The cardiometabolic protective action of the ANP gene variant rs5068 was also assessed. Subjects were followed for all-cause mortality and morbidity for 12 years. Results: Plasma ANP molecular forms were significantly lower in males compared to females. Males had higher BMI and greater incidence of myocardial infarction (MI), coronary artery disease and heart failure (HF). Males also had lower EF and HDL cholesterol and higher LA volume and LV mass, serum glucose, insulin and diastolic pressure. The presence of the rs5068 minor allele improved the cardiometabolic phenotype including BP and widespread metabolic parameters in males but not females. After adjustment by age, males had increased risk of all-cause mortality, HF, MI, and stroke. Conclusions: Males are characterized by a relative ANP deficiency compared to females and widespread prevalence of cardiometabolic disease and increased CV morbidity and mortality in the general population. We demonstrate that the cardiometabolic protective action of the ANP gene variant rs5068 minor allele is sex dependent and protective in males. These findings have diagnostic and therapeutic implications, particularly in males.

Introduction: Blood pressure (BP) control remains low despite combination therapy in resistant hypertension (RH). Thus, there is an unmet need for novel strategies targeting RH. Recently we have reported that M-ANP, a novel Mayo engineered ANP-based peptide, possesses greater and more sustained biological actions compared to ANP and is more resistant to neprilysin degradation. The current study was designed to investigate for the first time the efficacy of a single daily subcutaneous (SQ) injection of M-ANP using a chronic canine model of hypertension induced by angiotensin II (Ang-II). Hypothesis: We hypothesize that a daily SQ M-ANP injection would have potent BP lowering and renal enhancing properties in our chronic hypertensive canine model. Methods: We induced hypertension in canines (n=5) by continuous Ang II (80 ng/kg/min) osmotic mini-pump administration for 24 days. Continuous 24 hour mean BP (MBP) was measured by telemetry and a 24 hour urine collection was used to assess urinary volume, sodium (Na) and cGMP, the second messenger for the ANP system. M-ANP was administered SQ for 5 days (10μg/kg/day). Data are mean±SE. ΨP<0.05 Ang II Day 16 vs. baseline; *P<0.05 Ang II Day 16 vs. M-ANP (Table 1). Results: Chronic Ang II administration resulted in model of severe hypertension, with an increase in MBP from 112±2 to 157±2 mmHg (day 16). As seen in Table 1, M-ANP significantly reduced MBP, significantly increased plasma cGMP as well as urinary cGMP excretion, urinary volume rate (UV) and urinary Na excretion (UNa), without significant changes in heart rate (HR). Furthermore, there was a trend for plasma ANP to increase and plasma aldosterone to decrease with SQ M-ANP injection. Conclusions: This study demonstrates for the first time that chronic SQ administration of M-ANP reduces BP, activates plasma cGMP, and increases UV and UNa, without significant changes in HR in an experimental model of chronic hypertension.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones secreted as compensatory measures to preserve cardiorenal homeostasis. Treating heart failure (HF) with sacubitril/valsartan increased ANP and not BNP, suggesting the ANP system may confer therapeutic benefit. Thus, the presence of an ANP deficient subgroup of HF patients possibly posing higher risk for HF severity, underscores a need to characterize both, the biologically active and inactive (NT-proANP and NT-proBNP) natriuretic peptides (NPs)(latter resistant to degradation), to highlight the extent of NP production in HF. Characterizing neprilysin (NEP) activity may also provide insights into the degree of NP degradation in HF. This study aims to both highlight the cardiac NP response in acute decompensated HF(ADHF) by measuring molecular forms of ANP and BNP together with defining circulating soluble NEP activity as to assess mechanisms impairing the NP response in HF. We recruited 47 healthy individuals with neither cardiovascular nor metabolic disease and compared ANP, BNP, NT-proANP, NT-proBNP, and NEP activity levels with 107 recruited ADHF patients. In ADHF, compared to a healthy setting, median ANP, BNP, NT-proANP, and NT-proBNP were all elevated (15.7 vs. 118.1 pg/mL , p<0.0001; 23 vs. 515 pg/ml, p<0.0001; 60.1 vs. 651.6 pg/mL, p<0.0001; 46 vs. 3330 pg/mL, p<0.0001, respectively). However, circulating NEP activity was significantly lower in ADHF than in healthy circulation (32 vs. 12.3 nM/mL/min, p<0.0001). NPs did not correlate with NEP activity in healthy circulation. However, only ANP (r = 0.4788, p<0.0001) and NT-proANP (r = 0.6819, p<0.0001) correlated with NEP activity in ADHF. The presence of an impaired ANP response in ADHF, underscored by insufficient increases in NT-proANP than NT-proBNP, suggests decreased ANP production may induce not only an ANP deficient ADHF state, but be characteristic of ADHF. The presence of NEP activity in ADHF circulation, despite lower NEP activity compared to healthy individuals, suggests ANP degradation may second impaired production, further lowering ADHF ANP levels. Our studies support augmenting the ANP system in ADHF through either endogenous stimulation or exogenous supplementation as optimal therapeutic strategies.