Clearance Receptor Research Articles

A natriuretic peptides clearance receptor’s agonist reduces pulmonary artery pressures and enhances cardiac performance in preclinical models: New hope for patients with pulmonary hypertension due to left ventricular heart failure

BackgroundIn patients with left ventricular heart failure (HF), the development of pulmonary hypertension (PH) is common and represents a strong predictor of death. Despite recent advances in the pathophysiological understanding there is as yet no prospect of cure of this deadly clinical entity and the majority of patients continue to progress to right ventricular failure and die. Furthermore, there is no single medical treatment currently approved for PH related to HF. There is, therefore an urgent unmet need to identify novel pharmacological agents that will prevent the progressive increased or reverse the elevated pulmonary arterial pressures while enhancing cardiac performance in HF. Method and resultsWe here reported, for the first time, using a pressure-loop (P-V) conductance catheter system, that a specific natriuretic peptides clearance receptors’ agonist, the ring-deleted atrial natriuretic peptide analogue, cANF4-23 (cANF) reduces pulmonary artery pressures. Strikingly, the administration of the cANF in these mice decreased the RVSP by 50% (n=5, F 25.687, DF 14, p<0.001) and heart rate (HR) by 11% (n=5, F 25.69, DF 14, p<0.001) as well as enhancing cardiac performance including left ventricular contractility in mice. Most strikingly, mice lacking NPR-C were much more susceptible to develop HF, indicating that NPR-C is a critical protective receptor in the heart. ConclusionNatriuretic peptides clearance receptors’ agonists may, therefore represent a novel and attractive therapeutic strategy for PH related to HF, and ultimately improves the life expectancy and quality for millions of people around the planet.

Potential Role of Endothelin in Early Vascular Aging.

Early vascular aging is a process associated with gradual alterations in the vessels, regarding their structure and function, taking a more rapid course than normal biological aging in the arteries. In the presence of cardiovascular disease, these age-associated alterations are accelerated, contributing in the appearance or the progression of cardiovascular disease, such as high blood pressure, dyslipidemia, smoking and diabetes. Endothelin-1 (ET-1) is the most abundant and important endothelin produced by vascular cells. ET-1 exerts its biological actions through the activation of two receptors: ETA and ETB. Many important functions are mediated by the activation of these receptors, such as cardiovascular remodeling, vasoconstriction, cell proliferation and differentiation, production of extracellular matrix, and water and sodium secretion control. ETA receptor seems to participate in the pathogenesis and development of diseases, such as diabetes, atherosclerosis, systemic and pulmonary hypertension, and cardiac remodeling after myocardial ischemia, whereas ETB receptor seems to prevent the overstimulation of ETA receptor, acting as a clearance receptor. Increased ET-1 system activity may contribute to vascular dysfunction in aging via multiple pathways, such as direct hemodynamic effects, vascular oxidative stress, inflammatory activity, mitogenic stimulation of the vascular smooth muscle cells and fibrotic processes. Endothelin receptor antagonists were considered to be used for the treatment of some diseases like hypertension, diabetes and chronic kidney disease. However, besides pulmonary hypertension, this class is not in clinical use because of the side effects and the availability of safer drugs for the treatment of these diseases.

Modulation of natriuretic peptide receptors in human adipose tissue: molecular mechanisms behind the “natriuretic handicap” in morbidly obese patients

The B-type natriuretic peptide (BNP) hormone plays a crucial role in the regulation of cardiovascular and energy homeostasis. Obesity is associated with lowcirculating levels of BNP, a condition known as "natriuretic handicap." Recent evidences suggest an altered expression of BNP receptors-both the signaling natriuretic peptide receptors (NPR)-A and the clearance NPR-C receptor-in adipose tissue (AT) as one of the putative causes of natriuretic handicap. The current study aims at clarifying the molecular mechanisms behind the natriuretic handicap, focusing on NPR modulation in the AT of obese and control subjects. The study enrolled 34 obese and 20 control subjects undergoing bariatric or abdominal surgery, respectively. The main clinical and biochemical parameters, including circulating BNP, were assessed. In visceral (VAT) and subcutaneous AT (SAT) samples, collected during surgery, the adipocytes and stromal vascular fraction (SVF) expression of NPR-A and NPR-C and the SVF secretion of interleukin 6 (IL-6) were determined. Both VAT and SAT from obese patients expressed a lower NPR-A/NPR-C ratio in adipocytes and the SVF secreted a higher level of IL-6, compared with the controls. Moreover, NPR-A/NPR-C ratio expressed by VAT and SAT adipocytes negatively correlated with body mass index, insulin, the Homeostasis Model Assessment of Insulin resistance, and IL-6 secreted by SVF, and the expression of the clearance receptor NPR-C, in both the VAT and SAT adipocytes, showed a negative correlation with circulating BNP. Overall, insulin resistance/hyperinsulinemia and AT inflammation (ie, high level of IL-6) are the major determinants of the lower NPR-A/NPR-C ratio in adipocytes, thus contributing to the natriuretic handicap in obese subjects.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

Lipoprotein(a) Mass Levels Increase Significantly According to APOE Genotype: An Analysis of 431 239 Patients.

Lipoprotein(a) [Lp(a)] levels are genetically determined by hepatocyte apolipoprotein(a) synthesis, but catabolic pathways also influence circulating levels. APOE genotypes have different affinities for the low-density lipoprotein (LDL) receptor and LDL-related protein-1, with ε2 having the weakest binding to LDL receptor at <2% relative to ε3 and ε4. APPROACH AND RESULTS: APOE genotypes (ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, and ε4/ε4), Lp(a) mass, directly measured Lp(a)-cholesterol levels, and a variety of apoB-related lipoproteins were measured in 431 239 patients. The prevalence of APOE traits were ε2: 7.35%, ε3: 77.56%, and ε4: 15.09%. Mean (SD) Lp(a) levels were 65% higher in ε4/ε4 compared with ε2/ε2 genotypes and increased significantly according to APOE genotype: ε2/ε2: 23.4 (29.2), ε2/ε3: 31.3 (38.0), ε2/ε4: 32.8 (38.5), ε3/ε3: 33.2 (39.1), ε3/ε4: 35.5 (41.6), and ε4/ε4: 38.5 (44.1) mg/dL (P<0.0001). LDL-cholesterol, apoB, Lp(a)-cholesterol, LDL-cholesterol corrected for Lp(a)-cholesterol content, LDL-particle number, and small, dense LDL also had similar patterns. Patients with LDL-cholesterol ≥250 mg/dL, who are more likely to have LDL receptor mutations and reduced affinity for apoB, had higher Lp(a) levels across all apoE isoforms, but particularly in patients with ε2 alleles, compared with LDL <250 mg/dL. The lowest Lp(a) mass levels were present in patients with ε2 isoforms and lowest LDL-cholesterol. APOE genotypes strongly influence Lp(a) and apoB-related lipoprotein levels. This suggests that differences in affinity of apoE proteins for lipoprotein clearance receptors may affect Lp(a) catabolism, suggesting a competition between Lp(a) and apoE protein for similar receptors.

Von Willebrand Factor Interaction with FVIII: Development of Long Acting FVIII Therapies

Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) both play a centrol role in hemostasis, illustrated by the severe bleeding disorders associated with their functional absence. Despite their different functionalities in hemostasis and being products from two different genes, both proteins circulate in a tight, non-covalently linked complex. The physiological concequences of complex formation are many, including stabilization of FVIII heterodimeric structure, protection of FVIII from protelytic degradation, and modulation of FVIII immunogenicity.Another relevant issue relates to the chaperone function of VWF, allowing FVIII to survive in the circulation. FVIII levels are markedly reduced in patients with no detectable VWF protein or with a defect in VWF-FVIII complex formation, indicating that VWF prevents FVIII from premature clearance. Moreover, evidence points to FVIII actually being predominantly cleared as part of the VWF-FVIII complex rather than as a separate protein. First, it is possible to predict FVIII half-life fairly accurately by knowing antigen levels of VWF and its propeptide in combination with blood group. Second, when FVIII and VWF are co-injected in Vwf-deficient mice, FVIII is targeted to the same macrophages as is VWF.Since the end of the 1990s, our knowledge on the clearance mechanism of FVIII and VWF has started to emerge, and multiple clearance receptors for both proteins have now been identified. Interestingly, there exists a large overlap in receptor-repertoire between FVIII and VWF. These findings have taught us that it will be difficult to design single-mutant FVIII or VWF variants that have prolonged half-lives.How then to prolong the half-life of FVIII to improve treatment of hemophilia A? Several novel bioengineered FVIII variants have been developed, including PEGylation, Fc fusion and single-chain design, aiming to increase FVIII half-life. These approaches have so far achieved only moderate increases in half-life (1.5- to 2-fold compared to marketed FVIII products), significantly less than when similar modifications are being applied to factor IX. Indeed, it seems as if in designing these FVIII variants, the role of the significant other in the complex has been overlooked, since FVIII clearance is principally determined by VWF.Could we instead use VWF as a tool to prolong half-life of FVIII? This option is actually limited by the nature of the interaction between VWF and FVIII. Although of high affinity, the interaction is characterized by high association- and dissociation-rates. Infusing FVIII in combination with long-acting VWF variants will therefore result in a rapid redistribution of FVIII to endogenous VWF, as has elegantly been shown by the group of Ginsburg. To overcome this limitation, we have designed a FVIII variant (FVIII-KB013bv) in which we have replaced the B-domain by a single-domain, llama-derived antibody fragment (nanobody) that recognizes the D'D3-region of VWF. Consequently, the dissociation-rate of the VWF/FVIII complex is reduced 100-fold. Preliminary studies revealed that FVIII-KB013bv has a two-fold prolonged half-life compared to FVIII, likely due to improved VWF binding properties. Combination of the FVIII-nanobody fusion protein with long-acting VWF variants is anticipated to prolong its half-life well beyond the limit of the current long-acting FVIII variants. DisclosuresLenting:NovoNordisk: Consultancy, Research Funding.

Identification of glycans on plasma-derived ADAMTS13

AbstractPatients suffering from acquired thrombotic thrombocytopenic purpura develop autoantibodies directed toward the plasma glycoprotein ADAMTS13. Here, we studied the glycan composition of plasma-derived ADAMTS13. Purified ADAMTS13 was reduced, alkylated, and processed into peptides with either trypsin or chymotrypsin. Glycopeptides were enriched using zwitterionic HILIC zip-tips and analyzed by tandem mass spectrometry employing higher-energy collision dissociation fragmentation. Upon detection of a diagnostic ion of a glycan fragment, electron transfer dissociation fragmentation was performed on the same precursor ion. The majority of N-linked glycans were of the complex type containing terminal sialic acids and fucose residues. A high mannose-containing glycan was attached to Asn614 in the spacer domain. Six O-linked glycans mostly terminating in sialic acid were found dispersed over ADAMTS13. Five O-linked glycans were attached to a Ser and one to Thr. All 6 O-linked glycans contained a terminal sialic acid. O-fucosylation is a common posttranslational modification of thrombospondin type 1 repeats. We identified 7 O-fucosylation sites in the thrombospondin (TSP) type 1 repeats. Unexpectedly, one additional O-fucosylation site was found in the disintegrin domain. This O-fucosylation site did not meet the proposed consensus sequence CSX(S/T)CG. C-mannosylation sites were identified in TSP1, linker TSP4-TSP5, and TSP8. Overall, our findings highlight the complexity of glycan modifications on ADAMTS13, which may have implications for its interaction with immune- or clearance receptors containing carbohydrate recognition domains.

SP 04-1 THE ROLE OF NATRIURETIC PEPTIDES IN THE PATHOGENESIS OF CARDIOVASCULAR DISEASES

SP 04-1 THE ROLE OF NATRIURETIC PEPTIDES IN THE PATHOGENESIS OF CARDIOVASCULAR DISEASES

PS 16-01 INSULIN AND GLUCOSE INDUCE NATRIURETIC PEPTIDE CLEARANCE RECEPTOR IN HUMAN ADIPOCYTES

Objective: Cardiac natriuretic peptides (NP) are involved in cardio-renal regulation but also in lipolysis and lipid metabolism. The signaling receptor NPRA and the clearance receptor NPRC mediate the response to NP that is largely dependent on the ratio between the two receptors. Lipolysis increases when NPRC is selectively downregulated by starving or very-low calorie diet. Insulin is an anti-lipolytic hormone and also induces sodium-retention, suggesting a possible functional link with NP. Therefore we examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association between NP receptors expression in visceral adipose tissue (VAT) and insulin, HOMA index and metabolic profiles in patients undergoing renal surgery. Design and method: Differentiated human adipocytes from VAT and SGBS cell line were treated with insulin in the presence of glucose. Further experiments in low-glucose media were performed to simulate in vivo starving conditions and to study glucose-insulin mediated genes involved in lipogenesis. Lipolysis was assessed by glycerol release assay. Fasting blood samples and VAT samples were taken from patients in the day of renal surgery. Results: We observed a potent insulin-mediated and glucose-dependent up-regulation of NPRC, through the PI3K pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium hampered the insulin effect, likely through the modulation of glucose flux genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, higher HOMA index and higher total, LDL, and non-HDL cholesterol. Conclusions: Insulin and glucose-dependent NPRC induction in adipocytes, especially in the contest of insulin resistance and increased insulin levels, might be a key factor linking hyperinsulinemia, metabolic syndrome and higher blood pressure.

Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance.

Serpins are a widely distributed family of high molecular mass protein proteinase inhibitors that can inhibit both serine and cysteine proteinases by a remarkable mechanism-based kinetic trapping of an acyl or thioacyl enzyme intermediate that involves massive conformational transformation. The trapping is based on distortion of the proteinase in the complex, with energy derived from the unique metastability of the active serpin. Serpins are the favoured inhibitors for regulation of proteinases in complex proteolytic cascades, such as are involved in blood coagulation, fibrinolysis and complement activation, by virtue of the ability to modulate their specificity and reactivity. Given their prominence as inhibitors, much work has been carried out to understand not only the mechanism of inhibition, but how it is fine-tuned, both spatially and temporally. The metastability of the active state raises the question of how serpins fold, whereas the misfolding of some serpin variants that leads to polymerization and pathologies of liver disease, emphysema and dementia makes it clinically important to understand how such polymerization might occur. Finally, since binding of serpins and their proteinase complexes, particularly plasminogen activator inhibitor-1 (PAI-1), to the clearance and signalling receptor LRP1 (low density lipoprotein receptor-related protein 1), may affect pathways linked to cell migration, angiogenesis, and tumour progression, it is important to understand the nature and specificity of binding. The current state of understanding of these areas is addressed here.

Abstract 414: Putative Role of MicroRNA-143 in Modulating Natriuretic Peptide Signaling via Down-regulation of the Expression of Natriuretic Peptide Receptor 3

Cardiac natriuretic peptides (NPs) play important roles in the regulation of intravascular blood volume and vascular tone. Among other clearance mechanisms, bio-active circulating NPs are removed by the clearance receptor, Natriuretic Peptide Receptor 3 (NPR3). We hypothesized that the level of NPR3 could be modulated by microRNAs (miRNAs) resulting in changes in the bioactivity of NPs. We have previously reported a cluster of miRNAs potentially regulating NPR3 expression. To extend these findings, expression of the microRNAs concerned was examined in multiple platforms, including plasma from a clinical heart failure cohort, in the rat myocardial infarction model, and in a human cardiac derived cell line subjected to hypoxic challenge. Results: miR-143 was up-regulated in peripheral blood in heart failure patients compared with controls. The binding of miR-143 to the 3’UTR of NPR3 m RNA was verified by luciferase reporter assay. Antagomir-based silencing of miR-143 enhanced NPR3 expression in human derived cardiac cells. Elevation of miR-143 and down-regulation of NPR3 levels were observed in hypoxia treated cells and in the myocardium from the rat myocardial infarction model. Taken together, these findings suggest miR-143 may be involved in the down-regulation of NPR3 which in turn may provide more cardiac protective bioactivity from NPs in heart failure, myocardial hypoxic stress and in myocardial infarction. In summary, NPR3 is negatively regulated by miR-143, pointing to the therapeutic potential of miR-143 to beneficially enhance NP responses.

Over-Expressed Pathogenic miRNAs in Alzheimer's Disease (AD) and Prion Disease (PrD) Drive Deficits in TREM2-Mediated Aβ42 Peptide Clearance.

One prominent and distinguishing feature of progressive, age-related neurological diseases such as Alzheimer’s disease (AD) and prion disease (PrD) is the gradual accumulation of amyloids into dense, insoluble end-stage protein aggregates. These polymorphic proteolipid lesions are known to contribute to immunogenic and inflammatory pathology in these insidious and fatal disorders of the human central nervous system (CNS). For example, the evolution of self-aggregating amyloid-beta (Aβ) peptides, such as the 42 amino acid Aβ42 peptide monomer into higher order aggregates are largely due to: (1) the inability of natural processes to clear them from the cellular environment; and/or (2) the overproduction of these amyloid monomers which rapidly mature into higher order oligomers, fibrils and insoluble, end-stage senile plaques. Cells of the CNS such as microglial (MG) cells have evolved essential homeostatic mechanisms to clear Aβ peptides to avoid their accumulation, however, when defective, these clearance mechanisms become overwhelmed and excessive deposition and aggregation of these amyloids result. This ‘Perspectives’ paper will highlight some emerging concepts on the up-regulation of an inducible microRNA-34a in AD and PrD that drives the down-regulation of the amyloid sensing- and clearance receptor protein TREM2 (the triggering receptor expressed in myeloid/microglial cells). The impairment of this inducible, miRNA-34a-regulated TREM2- and MG-cell based amyloid clearance mechanism may thereby contribute to the age-related amyloidogenesis associated with both AD and PrD.

Design and Modular Construction of a Polymeric Nanoparticle for Targeted Atherosclerosis Positron Emission Tomography Imaging: A Story of 25% (64)Cu-CANF-Comb.

To assess the physicochemical properties, pharmacokinetic profiles, and in vivo positron emission tomography (PET) imaging of natriuretic peptide clearance receptors (NPRC) expressed on atherosclerotic plaque of a series of targeted, polymeric nanoparticles. To control their structure, non-targeted and targeted polymeric (comb) nanoparticles, conjugated with various amounts of c-atrial natriuretic peptide (CANF, 0, 5, 10 and 25%), were synthesized by controlled and modular chemistry. In vivo pharmacokinetic evaluation of these nanoparticles was performed in wildtype (WT) C57BL/6 mice after (64)Cu radiolabeling. PET imaging was performed on an apolipoprotein E-deficient (ApoE(-/-)) mouse atherosclerosis model to assess the NPRC targeting efficiency. For comparison, an in vivo blood metabolism study was carried out in WT mice. All three (64)Cu-CANF-comb nanoparticles showed improved biodistribution profiles, including significantly reduced accumulation in both liver and spleen, compared to the non-targeted (64)Cu-comb. Of the three nanoparticles, the 25% (64)Cu-CANF-comb demonstrated the best NPRC targeting specificity and sensitivity in ApoE(-/-) mice. Metabolism studies showed that the radiolabeled CANF-comb was stable in blood up to 9days. Histopathological analyses confirmed the up-regulation of NPRC along the progression of atherosclerosis. The 25% (64)Cu-CANF-comb demonstrated its potential as a PET imaging agent to detect atherosclerosis progression and status.

Transient silencing of Npr3 gene expression improved the circulatory levels of atrial natriuretic peptides and attenuated β-adrenoceptor activation- induced cardiac hypertrophic growth in experimental rats

Natriuretic peptide receptor-C (NPR-C) is considered as a clearance receptor that maintains the circulatory levels of natriuretic peptides. It has been suggested that augmented expression of NPR-C as a cause for the diminished anti-hypertrophic action of natriuretic peptides in the failing heart. Hence, we sought to determine the level of Npr3 gene (coding for NPR-C) expression in the Isoproterenol (ISO) treated Wistar rats. In addition, we studied the effect of Npr3 gene silencing on the hypertrophic growth. A significant increase in heart weight-to-body weight ratio (HW/BW-24%,P<0.01), an indicator of cardiac hypertrophic growth was observed in the ISO (10mg/kg BW/day,i.p for 7 days) treated rats. As expected, the cardiac NPR-C protein expression was significantly increased by 4 fold as compared to control rats. In parallel, the circulatory atrial natriuretic peptide (ANP) level was significantly decreased (2 fold) in ISO treated rats. Upon treatment with siRNA-Npr3, a significant decrease in the cardiac NPR-C protein expression (70%,P<0.01), HW/BW ratio (70%,P<0.01) and hypertrophic marker genes (α-Sk, β-MHC, c-fos, P<0.01, respectively) mRNA expression were observed. Interestingly, the circulatory ANP level was increased by 1.5 fold in the siRNA-Npr3 treated rats as compared to ISO treated rats. Moreover, the cardiac collagen content, matrixmetalloprotinases-2 (MMP-2) and enzymatic antioxidant status (P<0.01, respectively) were found to be restored back to near normal upon siRNA-Npr3 treatment. Taken together, the results of this study indicates that specific down-regulation of Npr3 gene improves the circulatory levels of ANP and antioxidant system and there by attenuates the β-adrenoceptor over-activation mediated cardiac hypertrophic growth in experimental rats.

Insulin/glucose induces natriuretic peptide clearance receptor in human adipocytes: a metabolic link with the cardiac natriuretic pathway.

Cardiac natriuretic peptides (NP) are involved in cardiorenal regulation and in lipolysis. The NP activity is largely dependent on the ratio between the signaling receptor NPRA and the clearance receptor NPRC. Lipolysis increases when NPRC is reduced by starving or very-low-calorie diet. On the contrary, insulin is an antilipolytic hormone that increases sodium retention, suggesting a possible functional link with NP. We examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association of NP receptor expression in visceral adipose tissue (VAT) with metabolic profiles of patients undergoing renal surgery. Differentiated human adipocytes from VAT and Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte cell line were treated with insulin in the presence of high-glucose or low-glucose media to study NP receptors and insulin/glucose-regulated pathways. Fasting blood samples and VAT samples were taken from patients on the day of renal surgery. We observed a potent insulin-mediated and glucose-dependent upregulation of NPRC, through the phosphatidylinositol 3-kinase pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium, used to simulate in vivo starving conditions, hampered the insulin effect on NPRC through modulation of insulin/glucose-regulated pathways, allowing atrial natriuretic peptide to induce lipolysis and thermogenic genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, HOMA-IR, and atherogenic lipid levels. Insulin/glucose-dependent NPRC induction in adipocytes might be a key factor linking hyperinsulinemia, metabolic syndrome, and higher blood pressure by reducing NP effects on adipocytes.

In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog.

The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor-dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.

Impact of Superoxide Dismutase Mimetic AEOL 10150 on the Endothelin System of Fischer 344 Rats.

Endothelin-1 is a potent vasoconstrictor and mitogenic peptide involved in the regulation of vasomotor tone and maintenance of blood pressure. Oxidative stress activates the endothelin system, and is implicated in pulmonary and cardiovascular diseases including hypertension, congestive heart failure, and atherosclerosis. Superoxide dismutase mimetics designed with the aim of treating diseases that involve reactive oxygen species in their pathophysiology may exert a hypotensive effect, but effects on the endothelin system are unknown. Our objective was to determine the effect of the superoxide dismutase mimetic AEOL 10150 on the basal endothelin system in vivo. Male Fischer-344 rats were injected subcutaneously with 0, 2 or 5 mg/kg body weight of AEOL 10150 in saline. Plasma oxidative stress markers and endothelins (bigET-1, ET-1, ET-2, ET-3) as well as lung and heart endothelin/nitric oxide system gene expressions were measured using HPLC-Coularray, HPLC-Fluorescence and RT-PCR respectively. AEOL 10150 reduced (p<0.05) the circulating levels of isoprostane (-25%) and 3-nitrotyrosine (-50%) measured in plasma 2h and 24h after treatment, confirming delivery of a physiologically-relevant dose and the potent antioxidant activity of the drug. The reduction in markers of oxidative stress coincided with sustained 24h decrease (p<0.05) of plasma levels of ET-1 (-50%) and ET-3 (-10%). Expression of preproET-1 and endothelin converting enzyme-1 mRNA were not altered significantly in the lungs. However preproET-1 (not significant) and ECE-1 mRNA (p<0.05) were increased (10–25%) in the heart. Changes in the lungs included decrease (p<0.05) of mRNA for the ET-1 clearance receptor ETB and the vasoconstriction-signaling ETA receptor (-30%), and an early surge of inducible nitric oxide synthase expression followed by sustained decrease (-40% after 24 hours). The results indicate that interception of the endogenous physiological flux of reactive nitrogen species and reactive oxygen species in rats impacts the endothelin/nitric oxide system, supporting a homeostatic relationship between those systems.

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

Heart Failure With Preserved Ejection Fraction Induces Beiging in Adipose Tissue

Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there are no evidence-based therapies for HFpEF. Clinical studies suggest a relationship between obesity-associated dysfunctional adipose tissue (AT) and HFpEF. However, an apparent obesity paradox exists in some HF populations with a higher body mass index. We sought to determine whether HFpEF exerted effects on AT and investigated the involved mechanisms. Mice underwent d-aldosterone infusion, uninephrectomy, and were given 1% saline for 4 weeks. HFpEF mice developed hypertension, left ventricular hypertrophy, and diastolic dysfunction and had higher myocardial natriuretic peptide expression. Although body weights were similar in HFpEF and sham-operated mice, white AT was significantly smaller in HFpEF than in sham (epididymal AT, 7.59 versus 10.67 mg/g; inguinal AT, 6.34 versus 8.38 mg/g). These changes were associated with smaller adipocyte size and increased beiging markers (ucp-1, cidea, and eva) in white AT. Similar findings were seen in HFpEF induced by transverse aortic constriction. Increased activation of natriuretic peptide signaling was seen in white AT of HFpEF mice. The ratio of the signaling receptor, natriuretic peptide receptor type A, to the clearance receptor, nprc, was increased as was p38 mitogen-activated protein kinase activation. However, HFpEF mice failed to regulate body temperature during cold temperature exposure. In HFpEF, despite a larger brown AT mass (5.96 versus 4.50 mg/g), brown AT showed reduced activity with decreased uncoupling protein 1 (ucp-1), cell death-inducing DFFA-like effector a (cidea), and epithelial V-like antigen (eva) expression and decreased expression of lipolytic enzymes (hormone-sensitive lipase, lipoprotein lipase, and fatty acid binding protein 4) versus sham. These findings show that HFpEF is associated with beiging in white AT and with dysfunctional brown AT.