ABSTRACT
BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.
Subject(s)
Adenylyl Cyclases/deficiency , Aorta/enzymology , Aortic Diseases/prevention & control , Atherosclerosis/prevention & control , Cholesterol Ester Transfer Proteins/deficiency , Plaque, Atherosclerotic , Adenylyl Cyclases/genetics , Adiposity , Animals , Aorta/pathology , Aorta/physiopathology , Aortic Diseases/enzymology , Aortic Diseases/genetics , Aortic Diseases/pathology , Atherosclerosis/enzymology , Atherosclerosis/genetics , Atherosclerosis/pathology , Autonomic Nervous System/physiopathology , Biological Factors/metabolism , Cell Proliferation , Cholesterol Ester Transfer Proteins/genetics , Diet, High-Fat , Disease Models, Animal , Endothelial Cells/enzymology , Endothelial Cells/pathology , Lipids/blood , Lipolysis , Macrophages/enzymology , Macrophages/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/metabolism , Proprotein Convertase 9/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , Signal Transduction , Vasodilation , Weight GainABSTRACT
LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a homolog of the hyaluronan receptor CD44, and one of the most widely used markers of lymphatic endothelial cells in normal and tumor tissues. However, the physiologic role of LYVE-1 in the lymphatic system still remains unclear. It is well established that fibroblast growth factor 2 (FGF2) induces lymphangiogenesis. Based on the known interaction between FGF2 and CD44 and based on the structural similarity of CD44 and LYVE-1, we investigated whether FGF2 might interact with LYVE-1. We found that FGF2 is able to bind LYVE-1 using AlphaScreen, or after surface-immobilization or in solution. FGF2 binds to LYVE-1 with a higher affinity than any other known LYVE-1binding molecules, such as hyaluronan or PDGF-BB. Glycosylation of LYVE-1 is important for FGF2 binding. Furthermore, FGF2 interacts with LYVE-1 when overexpressed in CHO cells. Soluble LYVE-1 and knockdown of LYVE-1 in lymphatic endothelial cells impaired FGF2 signaling and functions. In addition, FGF2 but not VEGF-C-induced in vivo lymphangiogenesis, was also inhibited. Conversely, FGF2 also modulates LYVE-1 expression in cells and ex vivo. Thus, our data demonstrate a functional relationship to the interaction between FGF2 and LYVE-1.
Subject(s)
Endothelial Cells/metabolism , Fibroblast Growth Factor 2/metabolism , Vesicular Transport Proteins/metabolism , Animals , Biomarkers/metabolism , CHO Cells , Cell Line , Cricetinae , Cricetulus , Endothelial Cells/drug effects , Fibroblast Growth Factor 2/administration & dosage , Fibroblast Growth Factor 2/genetics , Human Umbilical Vein Endothelial Cells , Humans , Hyaluronan Receptors/metabolism , Lymphangiogenesis/drug effects , Lymphangiogenesis/physiology , Mice , Mice, Inbred BALB C , Protein Interaction Maps , RNA, Small Interfering/genetics , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vesicular Transport Proteins/administration & dosage , Vesicular Transport Proteins/antagonists & inhibitors , Vesicular Transport Proteins/geneticsABSTRACT
PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.
Subject(s)
Cell Movement/physiology , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 12/metabolism , Amino Acid Substitution , Animals , Cells, Cultured , Embryo, Mammalian/cytology , Fibroblasts/cytology , Intracellular Signaling Peptides and Proteins/genetics , Mice, Knockout , Mutation, Missense , Protein Tyrosine Phosphatase, Non-Receptor Type 12/genetics , Two-Hybrid System Techniques , src Homology DomainsABSTRACT
Despite the best pharmacologic tools available, cardiovascular diseases (CVDs) remain a major cause of morbidity and mortality in developed countries. After 2 decades of research, new therapeutic targets, such as angiopoietin-like proteins (ANGPTLs), are emerging. ANGPTLs belong to a family of 8 members, from ANGPTL1 to ANGPTL8; they have structural homology with angiopoietins and are secreted in the circulation. ANGPTLs display a multitude of physiological and pathologic functions; they contribute to inflammation, angiogenesis, cell death, senescence, hematopoiesis, and play a role in repair, maintenance, and tissue homeostasis. ANGPTLs-particularly the triad ANGPTL3, 4, and 8-have an established role in lipid metabolism through the regulation of triacylglycerol trafficking according to the nutritional status. Some ANGPTLs also contribute to glucose metabolism. Therefore, dysregulation in ANGPTL expression associated with abnormal circulating levels are linked to a plethora of CVD and metabolic disorders including atherosclerosis, heart diseases, diabetes, but also obesity and cancers. Because ANGPTLs bind to different receptors according to the cell type, antagonists are therapeutically inadequate. Recently, direct inhibitors of ANGPTLs, mainly ANGPTL3, have been developed, and specific monoclonal antibodies and antisense oligonucleotides are currently being tested in clinical trials. The aim of the current review is to provide an up-to-date preclinical and clinical overview on the function of the 8 members of the ANGPTL family in the cardiovascular system, their contribution to CVD, and the therapeutic potential of manipulating some of them.
Subject(s)
Cardiovascular Diseases , Cardiovascular System , Peptide Hormones , Humans , Angiopoietin-like Proteins , Cardiovascular Diseases/prevention & control , Cardiovascular Diseases/drug therapy , Obesity , Biology , Angiopoietins/metabolism , Angiopoietins/therapeutic use , Angiopoietin-Like Protein 3 , Angiopoietin-Like Protein 8 , Peptide Hormones/therapeutic useABSTRACT
AIMS: The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. METHODS AND RESULTS: We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. CONCLUSION: Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.
Subject(s)
Adenylyl Cyclases , Atherosclerosis , Animals , Humans , Mice , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Atherosclerosis/genetics , Atherosclerosis/prevention & control , Atherosclerosis/metabolism , Colforsin/pharmacology , Colforsin/metabolism , Endothelial Cells/metabolism , Endothelium/metabolism , Thrombin/metabolism , Cyclic AMP/metabolismABSTRACT
Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.