Thomas Langenickel

Increased expression of renal neutral endopeptidase in severe heart failure.

The enzyme neutral endopeptidase (NEP; EC 3.4.24.11) cleaves several vasoactive peptides such as the atrial natriuretic peptide (ANP). ANP is a hormone of cardiac origin with diuretic and natriuretic actions. Despite elevated circulating levels of ANP, congestive heart failure (CHF) is characterized by progressive sodium and water retention. In order to elucidate the loss of natriuretic and diuretic properties of ANP in CHF we analyzed activity, protein concentrations, mRNA and immunostaining of NEP in kidneys of different models of severe CHF in the rat.CHF was induced by either aortocaval shunt, aortic banding or myocardial infarction in the rat. All models were defined by increased left ventricular end-diastolic pressure and decreased contractility. The diminished effectiveness of ANP was reflected by reduced cGMP/ANP ratio in animals with shunt or infarction. Renal NEP activity was increased in rats with aortocaval shunt (203 +/- 7%, p < 0.001), aortic banding (184 +/- 11%, p < 0.001) and infarction (149 +/- 10%, p < 0.005). Western blot analysis revealed a significant increase in renal NEP protein content in two models of CHF (shunt: 214 +/- 57%, p < 0.05; infarction: 310 +/- 53 %, p < 0.01). The elevated protein expression was paralleled by a threefold increase in renal NEP-mRNA level in the infarction model. The increased renal NEP protein expression and activity may lead to enhanced degradation of ANP and may contribute to the decreased renal response to ANP in heart failure. Thus, the capacity to counteract sodium and water retention, would be diminished. The increased renal NEP activity may therefore be a hitherto unknown factor in the progression of CHF.

Natriuretic peptide receptor B signaling in the cardiovascular system : protection from cardiac hypertrophy

Natriuretic peptides (NP) represent a family of structurally homologous but genetically distinct peptide hormones involved in regulation of fluid and electrolyte balance, blood pressure, fat metabolism, cell proliferation, and long bone growth. Recent work suggests a role for natriuretic peptide receptor B (NPR-B) signaling in regulation of cardiac growth by either a direct effect on cardiomyocytes or by modulation of other signaling pathways including the autonomic nervous system. The research links NPR-B for the first time to a cardiac phenotype in vivo and underlines the importance of the NP in the cardiovascular system. This manuscript will focus on the role of NPR-B and its ligand C-type natriuretic peptide in cardiovascular physiology and disease and will evaluate these new findings in the context of the known function of this receptor, with a perspective on how future research might further elucidate NPR-B function.

Perivascular T-Cell Infiltration Leads to Sustained Pulmonary Artery Remodeling after Endothelial Cell Damage

Pulmonary hypertension is a vascular proliferative disease characterized by pulmonary artery remodeling because of dysregulated endothelial and smooth muscle cell proliferation. Although the role of inflammation in the development of the disease is not well-defined, plexogenic lesions in human disease are characterized by perivascular inflammation composed, in part, of T cells. We explored the role of T-cell infiltration on pulmonary vascular remodeling after endothelial cell damage. We induced endothelial cell damage using monocrotaline and isolated the role of T cells by using Rag1(tm1Mom) mice and performing adoptive T-cell transfer. We found that monocrotaline causes pulmonary vascular endothelial cell injury followed by a perivascular inflammatory response. The infiltration of inflammatory cells primarily involves CD4(+) T cells and leads to the progressive muscularization of small (<30 μm) arterioles. Pulmonary vascular proliferative changes were accompanied by progressive and persistent elevations in right ventricular pressure and right ventricular hypertrophy. Supporting the central role of CD4(+) T cells in the inflammatory response, Rag1(tm1Mom) (Rag1(-/-)) mice, which are devoid of T and B cells, were protected from the development of vascular injury when exposed to monocrotaline. The introduction of T cells from control mice into Rag1(-/-) mice reproduced the vascular injury phenotype. These data indicate that after endothelial cell damage, CD4(+) T-cell infiltration participates in pulmonary vascular remodeling. This finding suggests that a CD4(+) T-cell immune response may contribute to the pathogenesis of inflammatory vascular lesions seen in some forms of pulmonary hypertension.

KIS protects against adverse vascular remodeling by opposing stathmin-mediated VSMC migration in mice

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.

Effects of Sacubitril/Valsartan (LCZ696) on Natriuresis, Diuresis, Blood Pressures, and NT-proBNP in Salt-Sensitive Hypertension.

Salt-sensitive hypertension (SSH) is characterized by impaired sodium excretion and subnormal vasodilatory response to salt loading. Sacubitril/valsartan (LCZ696) was hypothesized to increase natriuresis and diuresis and result in superior blood pressure control compared with valsartan in Asian patients with SSH. In this randomized, double-blind, crossover study, 72 patients with SSH received sacubitril/valsartan 400 mg and valsartan 320 mg once daily for 4 weeks each. SSH was diagnosed if the mean arterial pressure increased by ≥10% when patients switched from low (50 mmol/d) to high (320 mmol/d) sodium diet. The primary outcome was cumulative 6- and 24-hour sodium excretion after first dose administration. Compared with valsartan, sacubitril/valsartan was associated with a significant increase in natriuresis (adjusted treatment difference: 24.5 mmol/6 hours, 50.3 mmol/24 hours, both P<0.001) and diuresis (adjusted treatment difference: 291.2 mL/6 hours, P<0.001; 356.4 mL/24 hours, P=0.002) on day 1, but not on day 28, and greater reductions in office and ambulatory blood pressure on day 28. Despite morning dosing of both drugs, ambulatory blood pressure reductions were more pronounced at nighttime than at daytime or the 24-hour average. Compared with valsartan, sacubitril/valsartan significantly reduced N-terminal pro B-type natriuretic peptide levels on day 28 (adjusted treatment difference: −20%; P=0.001). Sacubitril/valsartan and valsartan were safe and well tolerated with no significant changes in body weight or serum sodium and potassium levels with either treatments. In conclusion, sacubitril/valsartan compared with valsartan was associated with short-term increases in natriuresis and diuresis, superior office and ambulatory blood pressure control, and significantly reduced N-terminal pro B-type natriuretic peptide levels in Asian patients with SSH. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01681576.

Forced Homodimerization by Site-Directed Mutagenesis Alters Guanylyl Cyclase Activity of Natriuretic Peptide Receptor B

Abstract—Natriuretic peptides mediate their physiologic effects through activation of membrane-bound, guanylyl cyclase–coupled receptors (NPRs). Receptor dimerization is an important feature of signal transduction. This study was aimed at characterizing structurally important residues of the extracellular ligand-binding domain of NPR-B for receptor dimerization and cGMP generation. Deletion mutagenesis was used to replace cysteine residues at positions 53 (C53S), 417 (C417S), and 426 (C426S) by serine. Receptor expression, dimerization, whole-cell cGMP response, and guanylyl cyclase activity of membrane fractions were determined in stably transfected COS-7 cells. C53S, C417S, and C426S mutants were expressed and found to form disulfide-bridged covalent dimers. In contrast to NPR-B and C53S, C417S and C426S mutants displayed constitutive activity in whole cells (C417S, 146±12%, P <0.01; C426S, 153±7% of ligand-independent NPR-B cGMP generation, P <0.01). The cGMP response of C417S and C426S mutants in whole cells was dose dependent and ≈4 times lower than that in NPR-B, whereas it was blunted in C53S-transfected cells (1 &mgr;mol/L CNP, NPR-B 2868±436%; C53S, 206±16% of control, P <0.001 vs NPR-B, C417S, and C426S). Guanylyl cyclase assay in transfected cells confirmed the constitutive activity of C417S and C426S mutants. These data suggest that receptor dimerization by covalent disulfide bridges alters ligand-independent as well as ligand-dependent receptor activity. Localization of the crosslink in relation to the cell membrane is important for configuration of the extracellular domain and the consecutive signal transduction.

Visinin-like protein 1 regulates natriuretic peptide receptor B in the heart

Accumulating evidence indicates that Visinin-like protein-1 (VILIP-1), a member of the family of neuronal calcium sensor proteins (NCS), modulates a variety of processes in extra-neuronal tissues. In this study, we describe VILIP-1 expression in the human heart, rat cardiomyocytes, and H9c2 cells, and demonstrate that VILIP-1 regulates the cell surface localization of natriuretic peptide receptor B (NPR-B). In preparations from failing hearts, we observed VILIP-1 downregulation and reduced NPR-B signalling. In conclusion, VILIP-1 deficiency may be responsible for the reduced efficiency of the natriuretic peptide system in cardiac hypertrophy and heart failure and may therefore serve as pharmacological target.

GA-binding protein regulates KIS gene expression, cell migration, and cell cycle progression

The cyclin‐dependent kinase inhibitor p27Kip1 arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/ threonine residues. Recently, we identified the serine/ threonine kinase, KIS, which phosphorylates p27Kip1 on serine 10 leading to nuclear export of p27Kip1 and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site ~116 bp 5′ to the translation start site, and sequences extending to ‐141 were sufficient for maximal promoter activity. Mutation in either of two Ets‐binding sites in this region resulted in an approximately 75–80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA‐binding protein (GABP). Knocking down GABPα by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPα‐deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27Kip1 protein levels and a decreased percentage of cells in S‐phase. Consistent with these findings, following vascular injury in vivo, GABPα‐heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum‐responsive GABP binding to Ets‐binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.— Crook M. F., Olive, M., Xue, H.‐H., Lange‐nickel T. H., Boehm, M., Leonard, W. J., Nabel E. G. GA‐binding protein regulates KIS gene expression, cell migration, and cell cycle progression. FASEB J. 22, 225–235 (2008)

Assessment of Drug Interaction Potential between LCZ696, an Angiotensin Receptor Neprilysin Inhibitor, and Digoxin or Warfa rin

LCZ696 (sacubitril/valsartan) is a first-in-class angiotensin receptor neprilysin inhibitor that simultaneously inhibits neprilysin and blocks the angiotensin II receptor. LCZ696 has been recently approved for treatment of HF and likely be co-administered with digoxin or warfarin. The drug interaction potential between LCZ696 and digoxin or warfarin was evaluated because of their potentially shared metabolic/elimination pathways. Two separate drug-drug interaction studies were conducted in healthy subjects: LCZ696 200 mg twice daily was co-administered with digoxin 0.25 mg once daily (n=24) and warfarin 25 mg single dose (n=26), respectively. The pharmacokinetic profiles of the LCZ696 analytes (sacubitril, LBQ657 and valsartan), digoxin and R- and S-warfarin, the pharmacodynamic effects of warfarin and the safety and tolerability of the investigational drugs were assessed. The geometric mean ratio (GMR) and 90%confidence interval (90% CI) for Cmax and AUCs of R- and S-warfarin, digoxin, and pharmacologically active LCZ696 analytes were within the bioequivalence range of 0.8-1.25 when co-administered. The GMR and 90% CI of warfarin pharmacodynamics effects were also within 0.8-1.25 range when co-administered with LCZ696. LCZ696 was generally safe and welltolerated when administered alone or in combination with digoxin/warfarin. No drug-drug interaction was observed upon co-administration of LCZ696 with digoxin/warfarin in healthy subjects.

Treatment with darusentan over 21 days improved cGMP generation in patients with chronic heart failure.

In heart failure, the cGMP to natriuretic peptide ratio is decreased and infusion of atrial natriuretic peptide (ANP) induces less cGMP generation. The ratio of the second messenger cGMP to plasma concentrations of ANP or brain natriuretic peptide (BNP) correlates with the effectiveness of natriuretic peptides. It was investigated whether blockade of the ET(A) receptor might improve the cGMP:NP ratio in heart failure. Patients with chronic heart failure (n=142; mean age=57 years) received oral treatment with the ET(A) antagonist darusentan (either 30, 100, 300 mg/day or placebo) on top of standard therapy over a period of 21 days in a randomized, double-blind, placebo-controlled, multicentre study. Plasma concentrations of ANP, BNP and cGMP were determined before randomization and after 21 days of treatment. In parallel with decreased pulmonary and systemic vascular resistance, 3 weeks of oral treatment with the ET(A) receptor antagonist darusentan reduced BNP plasma levels and increased the cGMP:BNP ratio significantly. The improved cGMP:BNP ratio might reflect the ability of chronic ET(A) receptor blockade to facilitate the generation of the second messenger cGMP, which points towards a favourable modulation of the natriuretic peptide effector system, in addition to haemodynamic improvement in heart failure patients.