ABSTRACT
Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.
Subject(s)
Aniline Compounds/pharmacology , Eye/blood supply , Receptor, TIE-2/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 3/metabolism , Retinal Vessels/pathology , Sulfonic Acids/pharmacology , Animals , Catalysis , Cell Hypoxia , Choroid/blood supply , Human Umbilical Vein Endothelial Cells , Humans , Hypoxia , Macular Degeneration , Mice , Mice, Transgenic , Oxygen/metabolism , Phosphorylation , Signal Transduction , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly selective and potent inhibitors of HPTPbeta. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadiazoles without a significant effect on HPTPbeta potency.
Subject(s)
Chemistry, Pharmaceutical/methods , Nerve Tissue Proteins/antagonists & inhibitors , Protein Tyrosine Phosphatases/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Hydrogen Bonding , Hydrolysis , Models, Chemical , Models, Molecular , Molecular Structure , Receptor-Like Protein Tyrosine Phosphatases, Class 5 , Structure-Activity RelationshipABSTRACT
High-throughput screening of the P&GP corporate repository against several protein tyrosine phosphatases identified the sulfamic acid moiety as potential phosphotyrosine mimetic. Incorporation of the sulfamic acid onto a 1,2,3,4-tetrahydroisoquinoline scaffold provided a promising starting point for PTP1B inhibitor design.
Subject(s)
Enzyme Inhibitors/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Sulfonic Acids/pharmacology , Tetrahydroisoquinolines/chemistry , Binding Sites , Binding, Competitive , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Ligands , Models, Molecular , Molecular Mimicry , Phosphotyrosine/metabolism , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistry , src Homology DomainsABSTRACT
OBJECTIVE: Studies have reported that administration of stromal cell-derived factor-1 (SDF-1), the ligand for the G-protein coupled receptor CXCR4, increased collateral blood flow in a mouse model of vascular insufficiency via recruitment of endothelial precursor cells (EPC). The present study investigated the contribution of mature endothelial cells in the actions of SDF-1. METHODS: The regulation of SDF-1 and CXCR4 was examined in the rat cornea cauterization (CC) and aortic ring (AR) model. The functional significance of the SDF-1/CXCR4 pathway was explored in cultured endothelial cells, the AR model, and on collateral blood flow in a rat model of vascular insufficiency. RESULTS: In the present study, the CXCR4 transcript was dramatically upregulated in the rat CC and AR explants, systems containing and lacking bone marrow-derived EPCs, respectively. Addition of AMD3100, a selective CXCR4 antagonist, had no effect on vessel growth in the AR alone, but completely inhibited SDF-1 mediated increases in vascular sprouting. In cultured endothelial cells, SDF-1 alone or in combination with vascular endothelial growth factor (VEGF) significantly enhanced cell survival and migration. Finally, systemic administration of SDF-1 in a rat model of arterial insufficiency enhanced collateral blood flow above vehicle control and equal to that of VEGF after 2 weeks of treatment. CONCLUSION: These studies support activation of the SDF-1/CXCR4 axis as a means to promote blood vessel growth and enhance collateral blood flow, at least in part, via direct effects on vascular endothelial cells.
Subject(s)
Chemokines, CXC/administration & dosage , Endothelium, Vascular/metabolism , Peripheral Vascular Diseases/drug therapy , Animals , Aorta , Biomarkers/analysis , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/genetics , Chemokines, CXC/therapeutic use , Collateral Circulation , Cornea/blood supply , Dose-Response Relationship, Drug , Endothelium, Vascular/pathology , Hindlimb/blood supply , Immunohistochemistry/methods , In Vitro Techniques , Models, Animal , Neovascularization, Pathologic , Oligonucleotide Array Sequence Analysis , Peripheral Vascular Diseases/metabolism , Peripheral Vascular Diseases/pathology , RNA, Messenger/analysis , Rats , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Regional Blood Flow/drug effectsABSTRACT
During embryonic development, the growth of blood vessels requires the coordinated activation of endothelial receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptor-2 (VEGFR-2) and Tie-2. Similarly, in adulthood, activation of endothelial RTKs has been shown to enhance development of the collateral circulation and improve blood flow to ischemic tissues. Recent evidence suggests that RTK activation is negatively regulated by protein tyrosine phosphatases (PTPs). In this study, we used the nonselective PTP inhibitor bis(maltolato)oxovanadium IV (BMOV) to test the potential efficacy of PTP inhibition as a means to enhance endothelial RTK activation and improve collateral blood flow. In cultured endothelial cells, pretreatment with BMOV augmented VEGFR-2 and Tie-2 tyrosine phosphorylation and enhanced VEGF- and angiopoietin-1-mediated cell survival. In rat aortic ring explants, BMOV enhanced vessel sprouting, a process that can be influenced by both VEGFR-2 and Tie-2 activation. Moreover, 2 wk of BMOV treatment in a rat model of peripheral vascular disease enhanced collateral blood flow similarly to VEGF, and after 4 wk, BMOV was superior to VEGF. Taken together, these studies provide evidence that PTPs are important regulators of endothelial RTK activation and for the first time demonstrate the potential utility of phosphatase inhibition as a means to promote collateral development and enhance collateral blood flow to ischemic tissue.
Subject(s)
Collateral Circulation , Protein Tyrosine Phosphatases/metabolism , Vascular Diseases/physiopathology , Animals , Cell Survival , Cells, Cultured , Collateral Circulation/drug effects , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Humans , In Vitro Techniques , Male , Neovascularization, Physiologic/drug effects , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Protein Tyrosine Phosphatases/antagonists & inhibitors , Pyrones/pharmacology , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, TIE-2/metabolism , Vanadates/pharmacology , Vascular Diseases/pathologyABSTRACT
Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO(4)) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds.
