Sangaralingham, S. Jeson

BACKGROUND: Cardiomyocyte oxidative stress significantly contributes to the progression of hypertension-induced heart failure, highlighting the need for targeted therapies. We developed a novel peptide, NPA7, that coactivates the GC-A (guanylyl cyclase A)/cGMP and MasR (Mas receptor)/cAMP pathway. This study aimed to test NPA7’s ability to inhibit oxidative stress by modulating the p62 (Sequestosome 1)-KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear factor erythroid 2-related factor 2) pathway in human cardiomyocytes (HCMs) and a rat model of hypertension. METHODS: Oxidative stress was induced in HCMs using H 2 O 2 with phosphate-buffered saline or NPA7 treatment. Intracellular reactive oxygen species levels were assessed via dihydroethidium staining. Western blotting analysis measured p62, KEAP1, and NRF2 protein levels, while GSH/GSSG (glutathione/glutathione disulfide) ratios and antioxidant gene expression were analyzed. HCMs were transfected with small interfering RNA targeting GC-A, MasR, or p62 before NPA7 and H 2 O 2 treatment. In vivo, spontaneously hypertensive rats received saline or NPA7, with normotensive Wistar Kyoto rats as control and cardiac oxidative stress, KEAP1 protein levels, NOX2 (NADPH oxidase 2), and p67 (NADPH oxidase subunit p67-phox) mRNA levels were measured. RESULTS: NPA7 reduced H 2 O 2 -induced reactive oxygen species levels and increased GSH/GSSG ratio in HCMs. Silencing GC-A (guanylyl cyclase A receptor) and MasR (Mas receptor) reversed NPA7’s effects. NPA7 activated the KEAP1-NRF2 pathway, enhancing NRF2’s antioxidant target gene expression. In p62 knockdown HCMs, NPA7-induced KEAP1 degradation and NRF2 activation were diminished. Reactive oxygen species levels were elevated in spontaneously hypertensive rat versusWistar Kyoto rats’ hearts, however, NPA7 treatment reduced myocardial reactive oxygen species, suppressed KEAP1 protein, and decreased NOX2 and p67 mRNA levels. CONCLUSIONS: NPA7 exhibits antioxidant properties in HCMs and spontaneously hypertensive rat hearts by targeting GC-A and MasR through the p62-KEAP1-NRF2 pathway, supporting a novel therapeutic approach against cardiovascular disease–related oxidative stress.

Abstract Background/Introduction MANP is a novel atrial natriuretic peptide (ANP) analog bioengineered as a particulate guanylyl cyclase A receptor (GC-A) activator with superior receptor binding, resistance to neprilysin degradation, natriuretic, aldosterone suppressing and blood pressure (BP) lowering properties than native ANP. MANP is now under clinical development for resistant hypertension (RH). The rationale for its design was the fundamental role of the ANP/GC-A system in BP homeostasis through which GC-A activation with MANP leads to BP reduction through renal, vascular, and hormonal mechanisms mediated by the second messenger cGMP. Purpose We investigated the safety, tolerability, cGMP activation and BP reductions of MANP compared to placebo in subjects with RH. Methods We performed a double-blind, placebo controlled randomized trial in 36 RH patients comprised of approximately 50% non-African Americans (NAA) and 50% AA. MANP was administered in 6 cohorts receiving ascending subcutaneous (SQ) doses of MANP once daily from 3 ug/kg to a maximal dose of 7 ug/kg or placebo for 5 days and then followed for 21 days after dosing. Endpoints included safety, cGMP activation and changes in systolic BP (SBP), aldosterone (Aldo) levels and glomerular filtration rate (GFR). Results MANP was safe and well tolerated. One patient in the 7 ug/kg cohort was discontinued due to symptomatic hypotension. Compared to baseline, plasma cGMP increased in all MANP groups on Day 1 and Day 5 with no change in the placebo group. The maximal SBP reduction in the MANP group on Days 1 (D1) and 5 (D5) was -17.5 (D1) and -16 mmHg (D5) with 3 ug/kg (lowest dose) and -39.5 (D1) and -36.3 mmHg (D5) with 7 ug/kg (highest dose); Changes in the Placebo group were -6.3 (D1) and -16.7 mmHg (D5). Notably, the SBP reductions in AA were greater than NAA in the 4 and 7 ug/kg cohorts. After the 1-hour timepoint on both the first and fifth days, Aldo and percentage of Aldo reduction generally remained suppressed compared to baseline. Twenty-one days after MANP dosing, SBP remained lower in three groups receiving SQ MANP (4, 4.5 and 7 ug/kg) compared to placebo in association with a preservation of GFR. There were no reported drug related serious adverse events. Conclusions This clinical trial demonstrates that once a day SQ administration of MANP in RH subjects activates cGMP, without attenuation over 5 days, and reduces SBP and preserves GFR. At higher doses of MANP, SBP reductions were greater in AA compared to NAA. MANP also demonstrated Aldo inhibiting actions compared to placebo. Importantly, MANP was overall safe and well-tolerated. Our findings support the continued clinical development of MANP as a novel GC-A activating peptide for the treatment of RH.

Abstract Myocardial fibrosis is a pathological hallmark of cardiovascular disease (CVD), and excessive fibrosis can lead to new-onset heart failure and increased mortality. Currently, pharmacological therapies for myocardial fibrosis are limited, highlighting the need for novel therapeutic approaches. The particulate guanylyl cyclase B (GC-B) receptor possesses beneficial antifibrotic actions through the binding of its natural ligand C-type natriuretic peptide (CNP) and the generation of the intracellular second messenger, cyclic guanosine 3′,5′-monophosphate (cGMP). These actions include the suppression of fibroblast proliferation and reduction in collagen synthesis. With its abundant expression on fibroblasts, the GC-B receptor has emerged as a key molecular target for innovative CVD therapeutics. However, small molecules that can bind and potentiate the GC-B/cGMP pathway have yet to be discovered. From a cell-based high-throughput screening initiative of the NIH Molecular Libraries Small Molecule Repository and hit-to-lead evolution based on a series of structure–activity relationships, we report the successful discovery of MCUF-42, a GC-B-targeted small molecule that acts as a positive allosteric modulator (PAM). Studies herein support MCUF-42's ability to enhance the binding affinity between GC-B and CNP. Moreover, MCUF-42 potentiated cGMP levels induced by CNP in human cardiac fibroblasts (HCFs) and notably also enhanced the inhibitory effect of CNP on HCF proliferation. Together, our findings highlight that MCUF-42 is a small molecule that can modulate the GC-B/cGMP signaling pathway, potentially enhancing the antifibrotic actions of CNP. Thus, these data underscore the continued development of GC-B small molecule PAMs as a novel therapeutic strategy for targeting cardiac fibrosis and CVD.

Introduction: Cardiorenal fibrotic remodeling (CFR), often in the setting of heart failure (HF) and kidney disease (CKD), is a major cause for increased risk in mortality, for which there are no effective drugs. C-type natriuretic peptide (CNP) is a 22 amino acid (AA) ligand for the particulate guanylyl cyclase receptor-B (GC-B) and exerts anti-fibrotic actions, with little effect on blood pressure (BP) or renal function, via its second messenger cGMP. To overcome CNP’s limited efficacy with its short half-life, we designed 2 novel CNP-based peptides (C53 and CU32). C53 consists of CNP plus a 31 AA N-terminus extension, while CU32 is the CNP ring fused with the N- and C-termini of urodilatin (GC-A agonist). Here we investigated the cGMP generating actions of C53 and CU32, with the goal of advancing novel GC-B/cGMP therapeutics for CFR. Hypotheses: We hypothesized that C53 and CU32 would have cGMP activating actions in vivo, however only C53 would generate cGMP production selectively via GC-B in vitro. We also hypothesize that the addition of the C-termini to CU32 would form a non-selective GC-B/cGMP agonist with renal and BP lowering actions. Methods: HEK293 cells expressing human GC-B or -A and human fibroblasts (hFs), where GC-B is abundant, were stimulated with C53 or CU32 (10-8M) for 10 min. Two groups of normal rats (n=8) received a 75 min equimolar IV infusion of C53 or CU32. Change in mean arterial pressure (ΔMAP), sodium (Na) excretion and plasma and urinary cGMP were assessed. *p<0.05. Results: C53 and CU32 activated cGMP in GC-B cells (C53: 75±9, CU32: 24±2 pmol/well*), while only CU32 activated cGMP in GC-A cells (C53: 0±0, CU32: 50±3 pmol/well*). In hFs, both C53 and CU32 generated cGMP. In vivo, plasma cGMP (C53: 91±4, CU32: 94±6 pmol/ml) and urinary cGMP excretion (C53: 177±28, CU32: 227±28 pmol/min) were similar with both peptides. Yet, CU32 induced a greater reduction in ΔMAP (C53: -7±1, CU32: -16±3 mmHg*) and increased Na excretion (C53: 1.4±0.2, CU32: 2.4±0.2 uEq/m*) compared to C53. Conclusions: We report the engineering of a selective (C53) and non-selective (CU32) GC-B agonist that has favorable cGMP generating action in hFs and in vivo. Thus, C53 and CU32 may represent novel GC-B therapeutics that could be tailored to specific phases of HF or CKD to prevent CFR.

Background: C-type natriuretic peptide (CNP) is of endothelial cell origin and represents the most potent anti-fibrotic peptide of the natriuretic peptide family and is important for extracellular matrix homeostasis including mediating bone growth in the young. Cardiorenal aging is associated with altered cardiac and renal structure and function that may contribute to the development of heart and kidney failure, particularly in the elderly. The impact of aging on circulating CNP and associated changes in cardiorenal structure and function are undefined. Hypothesis: We hypothesized that a decrease in endogenous plasma CNP occurs with aging and is associated with an increase in cardiorenal fibrosis and altered left ventricular (LV) and renal structure and function. Methods: Studies were performed in 2, 11 and 20 month old male Fischer rats (n = 8 –10/group). Echo was used to assess LV structure and function. Blood pressure (BP), urinary protein excretion, plasma B-type natriuretic peptide (BNP) and CNP were measured. Kidneys and LV were harvested for gross and histology analysis. Mean±SE, *p<0.05. Results: Aging from 2 to 20 months was associated with progressive reductions in plasma CNP (30±3 to 21±1* to 9±1*pg/ml). Significant and progressive cardiac and renal fibrosis were observed with aging (LV: 9±0.3 to 15±0.7* to 21±1*% and kidney: 1±0.3 to 8±2* to 17±1* %). In contrast to CNP, plasma BNP was modestly increased from the 2 to 20 month old group (21±2 to 26±1*pg/ml). The reduction in CNP and increase in BNP, the latter a biomarker for LV dysfunction, were associated with decreased EF (88±1 to 80±1*%) and increased LVEDd (6.61±0.09 to 7.48±0.09*mm). BP and urinary protein excretion were mildly elevated with aging (91±1 to 101±3* mmHg and 8±0.3 to 20±3* mg/day, respectively). Conclusion: We report for the first time that aging is associated with a progressive decline in circulating CNP and progressive cardiorenal fibrosis, systolic dysfunction and proteinuria. Further studies are warranted to explore the hypothesis that a mechanism of cardiorenal aging with altered cardiorenal structure and function may include a decrease in the bioavailability of CNP.

Males are characterized by lower ANP and BNP with greater prevalence of cardiometabolic disease. The ANP genetic variant rs5068 was associated with less metabolic dysfunction in males, whereas no metabolic profile was related to the BNP genetic variant rs198389 in the general population. ANP may play a more biological role in metabolic homeostasis compared with BNP in the general population with greater physiological metabolic actions in males compared with females.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII–NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Abstract The discovery of the heart as an endocrine organ resulted in a remarkable recognition of the natriuretic peptide system (NPS). Specifically, research has established the production of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) from the heart, which exert pleiotropic cardiovascular, endocrine, renal, and metabolic actions via the particulate guanylyl cyclase A receptor (GC-A) and the second messenger, cGMP. C-type natriuretic peptide (CNP) is produced in the endothelium and kidney and mediates important protective auto/paracrine actions via GC-B and cGMP. These actions, in part, participate in the efficacy of sacubitril/valsartan in heart failure (HF) due to the augmentation of the NPS. Here, we will review important insights into the biology of the NPS, the role of precision medicine, and focus on the phenotypes of human genetic variants of ANP and BNP in the general population and the relevance to HF. We will also provide an update of the existence of NP deficiency states, including in HF, which provide the rationale for further therapeutics for the NPS. Finally, we will review the field of peptide engineering and the development of novel designer NPs for the treatment of HF. Notably, the recent discovery of a first-in-class small molecule GC-A enhancer, which is orally deliverable, will be highlighted. These innovative designer NPs and small molecule possess enhanced and novel properties for the treatment of HF and cardiovascular diseases.