ABSTRACT
Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of alpha,alpha-difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF-induced angiogenesis. DFPMPA-3, the most effective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its effectiveness was proven through neo-vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy.
Subject(s)
Enzyme Inhibitors/pharmacology , Neovascularization, Physiologic/drug effects , Organophosphonates/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Animals , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Endothelial Growth Factors/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , Lymphokines/physiology , Mice , Mice, Inbred ICR , Neovascularization, Physiologic/physiology , Organophosphonates/chemical synthesis , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.
Subject(s)
Brain Ischemia/pathology , Enzyme Inhibitors/pharmacology , Neurons/enzymology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Animals , Blotting, Western , Caspase 3 , Caspases/metabolism , Cell Death/drug effects , Cell Differentiation/drug effects , Ceramides/biosynthesis , DNA/analysis , DNA/biosynthesis , DNA Fragmentation , Electrophoresis, Polyacrylamide Gel , Glucose/deficiency , Indicators and Reagents , Infarction, Middle Cerebral Artery/pathology , JNK Mitogen-Activated Protein Kinases , Male , Mice , Mitogen-Activated Protein Kinases/metabolism , Neurons/drug effects , PC12 Cells , Phosphorylation , RatsABSTRACT
A series of short-chain analogues of N-palmitoylsphingosine-1-phosphate, modified by replacement of the phosphate and the long alkenyl side chain with hydrolytically stable difluoromethylene phosphonate and phenyl, respectively, were prepared to study the structure-activity relationship for inhibition of sphingomyelinase. The study revealed that inhibition is highly dependent upon the stereochemistry of the asymmetric centers of the acylamino moiety, and resulted in identification of a non-competitive inhibitor with the same level of inhibitory activity of schyphostatin, the most potent of the few known small molecular inhibitors of sphingomyelinase.
Subject(s)
Ceramides/chemical synthesis , Ceramides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Phospholipids/chemical synthesis , Phospholipids/pharmacology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Animals , Bacillus/drug effects , Bacillus/enzymology , Binding, Competitive/drug effects , Dogs , In Vitro Techniques , Indicators and Reagents , Kinetics , Magnetic Resonance Spectroscopy , Microsomes/drug effects , Microsomes/enzymology , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Sphingomyelin (SM) pathway, where sphingomyelinase (SMase) hydrolyzes SM to produce ceramide, has recently been suggested to link to the signaling pathway that determines cell death. Therefore, elucidation of the mechanism by which SMase is activated and the regulation of SMase activity will be an important therapeutic strategy for various cytokine-related and ischemic diseases. We have synthesized nine difluoromethylene analogues of SM as SMase inhibitors and evaluated their inhibitory potencies. In this study, we show that the inhibitor suppresses ceramide production and cell death of PC-12 neurons that have been induced by deprivation of serum from the culture medium. The SMase inhibitor could also suppress neuronal cell death in a mouse model of brain ischemia. These results suggest a possibility that the prevention of various extracellular stress-induced cell death signalings is accomplished by inhibiting the cell membrane SMase.