Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease.

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.

Arterial Pulsatility and Circulating von Willebrand Factor in Patients on Mechanical Circulatory Support.

BACKGROUND: The main risk factor for bleeding in patients with continuous-flow mechanical circulatory support (CF-MCS) is the acquired von Willebrand factor (VWF) defect related to the high shear-stress forces developed by these devices. Although a higher bleeding rate has been reported in CF-MCS recipients who had reduced pulsatility, the relation between pulsatility and the VWF defect has never been studied. OBJECTIVES: The purpose of this study was to investigate the relation between pulsatility and VWF under CF-MCS. METHODS: We assessed the effect of 2 CF-MCS on VWF multimer degradation in a mock circulatory loop (model 1). Using these devices, we investigated in a dose-effect model (model 2) 3 levels of pulsatility in 3 groups of swine. In a cross-over model (model 3), we studied the effects of sequential changes of pulsatility on VWF. We reported the evolution of VWF multimerization in a patient undergoing serial CF-MCS and/or pulsatile-MCS. RESULTS: We demonstrated the proteolytic degradation of VWF multimers by high shear CF-MCS in a circulatory loop without pulsatility. We observed both in swine models and in a patient that the magnitude of the VWF degradation is modulated by the pulsatility level in the high shear-stress level condition, and that the restoration of pulsatility is a trigger for the endothelial release of VWF. CONCLUSIONS: We demonstrated that the VWF defect reflects the balance between degradation induced by the shear stress and the endothelial release of new VWF triggered by the pulsatility. This modulation of VWF levels could explain the relationship between pulsatility and bleeding observed in CF-MCS recipients. Preservation of pulsatility may be a new target to improve clinical outcomes of patients.

Regulation of the interaction between the neuronal BIN1 isoform 1 and Tau proteins - role of the SH3 domain.

Bridging integrator 1 (bin1) gene is a genetic determinant of Alzheimer's disease (AD) and has been reported to modulate Alzheimer's pathogenesis through pathway(s) involving Tau. The functional impact of Tau/BIN1 interaction as well as the molecular details of this interaction are still not fully resolved. As a consequence, how BIN1 through its interaction with Tau affects AD risk is also still not determined. To progress in this understanding, interaction of Tau with two BIN1 isoforms was investigated using Nuclear Magnetic Resonance spectroscopy. 1 H, 15 N spectra showed that the C-terminal SH3 domain of BIN1 isoform 1 (BIN1Iso1) is not mobile in solution but locked with the core of the protein. In contrast, the SH3 domain of BIN1 isoform 9 (BIN1Iso9) behaves as an independent mobile domain. This reveals an equilibrium between close and open conformations for the SH3 domain. Interestingly, a 334-376 peptide from the clathrin and AP-2-binding domain (CLAP) domain of BIN1Iso1, which contains a SH3-binding site, is able to compete with BIN1-SH3 intramolecular interaction. For both BIN1 isoforms, the SH3 domain can interact with Tau(210-240) sequence. Tau(210-240) peptide can indeed displace the intramolecular interaction of the BIN1-SH3 of BIN1Iso1 and form a complex with the released domain. The measured Kd were in agreement with a stronger affinity of Tau peptide. Both CLAP and Tau peptides occupied the same surface on the BIN1-SH3 domain, showing that their interaction is mutually exclusive. These results emphasize an additional level of complexity in the regulation of the interaction between BIN1 and Tau dependent of the BIN1 isoforms.

Leptin induces osteoblast differentiation of human valvular interstitial cells via the Akt and ERK pathways.

AIMS: Calcific aortic valve disease (CAVD) affects 2-6% of the population over 65 years, and age, gender, smoking, overweight, dyslipidemia, diabetes contribute to the development of this disease. CAVD results, in part, from the osteoblast differentiation of human valvular interstitial cells (VICs). This study aims to elucidate the effects of leptin on osteoblast phenotype of VICs and the signalling pathways involved. METHODS: Patients who underwent aortic valve replacement for CAVD (n = 43) were included in this study. Patients with coronary artery disease (CAD) without CAVD (n = 129) were used as controls. RESULTS: Patients with CAVD had higher serum leptin concentrations than CAD patients (p = 0.002). Leptin was found in calcific aortic valves, with higher concentrations in calcified versus non-calcified zones (p = 0.01). Chronic leptin stimulation of human VICs enhanced alkaline phosphatase (ALP) activity and ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. Moreover, inhibiting Akt or ERK during leptin stimulation lowered the expression of osteoblast markers in VIC. CONCLUSIONS: Taken together, these findings indicate that leptin plays a critical role in CAVD development by promoting osteoblast differentiation of human aortic VICs in an Akt- and ERK-dependent manner. This study highlights the role of leptin in CAVD development, and further studies are needed to determine whether reducing circulating leptin levels or blocking leptin actions on VICs is efficient to slow CAVD progression.

Ischemia/reperfusion-induced alterations of enzymatic and signaling functions of the rat cardiac Na+/K+-ATPase: protection by ouabain preconditioning.

Cardiac glycosides (CG) are traditionally known as positive cardiac inotropes that inhibit Na+/K+-ATPase-dependent ion transport. CG also trigger-specific signaling pathways through the cardiac Na+/K+-ATPase, with beneficial effects in ischemia/reperfusion (I/R) injury (e.g., ouabain preconditioning, known as OPC) and hypertrophy. Our current understanding of hypersensitivity to CG and subsequent toxicity in the ischemic heart is mostly based on specific I/R-induced alterations of the Na+/K+-ATPase enzymatic function and has remained incomplete. The primary goal of this study was to investigate and compare the impact of I/R on Na+/K+-ATPase enzymatic and signaling functions. Second, we assessed the impact of OPC on both functions. Langendorff-perfused rat hearts were exposed to 30 min of ischemia and 30 min of reperfusion. At the inotropic concentration of 50 µmol/L, ouabain increased ERK and Akt phosphorylation in control hearts. In I/R hearts, this concentration did not induced positive inotropy and failed to induce Akt or ERK phosphorylation. The inotropic response to dobutamine as well as insulin signaling persisted, suggesting specific alterations of Na+/K+-ATPase. Indeed, Na+/K+-ATPase protein expression was intact, but the enzyme activity was decreased by 60% and the enzymatic function of the isoform with high affinity for ouabain was abolished following I/R. Strikingly, OPC prevented all I/R-induced alterations of the receptor. Further studies are needed to reveal the respective roles of I/R-induced modulations of Na+/K+-ATPase enzymatic and signaling functions in cardiomyocyte death.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.

Tau phosphorylation regulates the interaction between BIN1's SH3 domain and Tau's proline-rich domain.

INTRODUCTION: The application of high-throughput genomic approaches has revealed 24 novel risk loci for Alzheimer's disease (AD). We recently reported that the bridging integrator 1 (BIN1) risk gene is linked to Tau pathology. RESULTS: We used glutathione S-transferase pull-down assays and nuclear magnetic resonance (NMR) experiments to demonstrate that BIN1 and Tau proteins interact directly and then map the interaction between BIN1's SH3 domain and Tau's proline-rich domain (PRD) . Our NMR data showed that Tau phosphorylation at Thr231 weakens the SH3-PRD interaction. Using primary neurons, we found that BIN1-Tau complexes partly co-localize with the actin cytoskeleton; however, these complexes were not observed with Thr231-phosphorylated Tau species. CONCLUSION: Our results show that (i) BIN1 and Tau bind through an SH3-PRD interaction and (ii) the interaction is downregulated by phosphorylation of Tau Thr231 (and potentially other residues). Our study sheds new light on regulation of the BIN1/Tau interaction and opens up new avenues for exploring its complex's role in the pathogenesis of AD.

Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning.

Na(+),K(+)-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na(+),K(+)-ATPase α(1)- and α(3)-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α(1)-cell surface abundance was significantly decreased. Na(+),K(+)-ATPase-activity in crude homogenates and (86)Rb(+) transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 µM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na(+),K(+)-ATPase activity and surface expression. This model also revealed that Na(+),K(+)-ATPase-mediated (86)Rb(+) uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na(+),K(+)-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na(+),K(+)-ATPase α(1) known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na(+),K(+)-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na(+),K(+)-ATPase may be independent of ion transport.

Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis.

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.

Modulation of Na(+)-K(+)-ATPase cell surface abundance through structural determinants on the α1-subunit.

Through their ion-pumping and non-ion-pumping functions, Na(+)-K(+)-ATPase protein complexes at the plasma membrane are critical to intracellular homeostasis and to the physiological and pharmacological actions of cardiotonic steroids. Alteration of the abundance of Na(+)-K(+)-ATPase units at the cell surface is one of the mechanisms for Na(+)-K(+)-ATPase regulation in health and diseases that has been closely examined over the past few decades. We here summarize these findings, with emphasis on studies that explicitly tested the involvement of defined regions or residues on the Na(+)-K(+)-ATPase α1 polypeptide. We also report new findings on the effect of manipulating Na(+)-K(+)-ATPase membrane abundance by targeting one of these defined regions: a dileucine motif of the form [D/E]XXXL[L/I]. In this study, opossum kidney cells stably expressing rat α1 Na(+)-K(+)-ATPase or a mutant where the motif was disrupted (α1-L499V) were exposed to 30 min of substrate/coverslip-induced-ischemia followed by reperfusion (I-R). Biotinylation studies suggested that I-R itself acted as an inducer of Na(+)-K(+)-ATPase internalization and that surface expression of the mutant was higher than the native Na(+)-K(+)-ATPase before and after ischemia. Annexin V/propidium iodide staining and lactate dehydrogenase release suggested that I-R injury was reduced in α1-L499V-expressing cells compared with α1-expressing cells. Hence, modulation of Na(+)-K(+)-ATPase cell surface abundance through structural determinants on the α-subunit is an important mechanism of regulation of cellular Na(+)-K(+)-ATPase in various physiological and pathophysiological conditions, with a significant impact on cell survival in face of an ischemic stress.

Critical role of the isoform-specific region in alpha1-Na,K-ATPase trafficking and protein Kinase C-dependent regulation.

The isoform-specific region (ISR) is a region of structural heterogeneity among the four isoforms of the catalytic alpha-subunit of the Na,K-ATPase and an important structural determinant for isoform-specific functions. In the present study, we examined the role of a potential dileucine clathrin adaptor recognition motif [DE]XXXL[LI] embedded within the alpha1-ISR. To this end, a rat alpha1 construct where leucine 499 was replaced by a valine (as found in the alpha2 isoform sequence) was compared to wild-type rat alpha1 after stable expression in opossum kidney cells. Total Na,K-ATPase expression, activity, or in situ (86)Rb(+) transport was not affected by the L499V mutation. However, surface Na,K-ATPase expression was nearly doubled. This increase was associated with a reduced rate of internalization from the cell surface of about 50% after a 4 h chase and became undetectable if clathrin-coated pit-mediated trafficking was blocked with chlorpromazine. Further, PKC-induced stimulation of Na,K-ATPase-mediated (86)Rb(+) uptake was doubled in mutant-expressing cells, comparable to the chimera containing the intact alpha2-ISR. Similar results were observed when the potential motif was disrupted by means of an E495S mutation. These findings suggest that a dileucine motif embedded within the Na,K-ATPase alpha1-ISR plays a critical role in the surface expression of Na,K-ATPase alpha1 polypeptides at steady state and in the response to PKC activation.

Isoform specificity of Na-K-ATPase-mediated ouabain signaling.

The ion transporter Na-K-ATPase functions as a cell signal transducer that mediates ouabain-induced activation of protein kinases, such as ERK. While Na-K-ATPase composed of the alpha(1)-polypeptide is involved in cell signaling, the role of other alpha-isoforms (alpha(2), alpha(3), and alpha(4)) in transmitting ouabain effects is unknown. We have explored this using baculovirus-directed expression of Na-K-ATPase polypeptides in insect cells and ERK phosphorylation as an indicator of ouabain-induced signaling. Ouabain addition to Sf-9 cells coexpressing Na-K-ATPase alpha(1)- and beta(1)-isoforms stimulated ERK phosphorylation. In contrast, expression of the alpha(1)- and beta(1)-polypeptides alone resulted in no effect, indicating that the alphabeta-complex is necessary for Na-K-ATPase signaling. Moreover, the ouabain effect was sensitive to genistein, suggesting that Na-K-ATPase-mediated tyrosine kinase activation is a critical event in the intracellular cascade leading to ERK phosphorylation. In addition, the Na-K-ATPases alpha(3)beta(1)- and alpha(4)beta(1)-isozymes, but not alpha(2)beta(1), responded to ouabain treatment. In agreement with the differences in ouabain affinity of the alpha-polypeptides, alpha(1)beta(1) required 100- to 1,000-fold more ouabain to signal than did alpha(4)beta(1) and alpha(3)beta(1), respectively. These results confirm the role of the Na-K-ATPase in ouabain signal transduction, show that there are important isoform-specific differences in Na-K-ATPase signaling, and demonstrate the suitability of the baculovirus expression system for studying Na-K-ATPase-mediated ouabain effects.