ABSTRACT
Compensatory angiogenesis is an important adaptation for recovery from critical ischemia. We recently identified 20-hydroxyeicosatetraenoic acid (20-HETE) as a novel contributor of ischemia-induced angiogenesis. However, the precise mechanisms by which ischemia promotes 20-HETE increases that drive angiogenesis are unknown. This study aims to address the hypothesis that inflammatory neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to 20-HETE increases leading to ischemic angiogenesis. Using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, Laser Doppler Perfusion Imaging, and Microvascular Density analysis, we found that neutrophil depletion and MPO knockout mitigate angiogenesis and 20-HETE production in the gracilis muscles of mice subjected to hindlimb ischemia. Furthermore, we found MPO and HOCl to be elevated in these tissues postischemia as assessed by immunofluorescence microscopy and in vivo live imaging of HOCl. Next, we demonstrated that the additions of either HOCl or an enzymatic system for generating HOCl to endothelial cells increase the expression of CYP4A11 and its product, 20-HETE. Finally, pharmacological interference of hypoxia inducible factor (HIF) signaling results in ablation of HOCl-induced CYP4A11 transcript and significant reductions in CYP4A11 protein. Collectively, we conclude that neutrophil-derived MPO and its product HOCl activate HIF-1α and CYP4A11 leading to increased 20-HETE production that drives postischemic compensatory angiogenesis. SIGNIFICANCE STATEMENT: Traditionally, neutrophil derived MPO and HOCl are exclusively associated in the innate immunity as potent bactericidal/virucidal factors. The present study establishes a novel paradigm by proposing a unique function for MPO/HOCl as signaling agents that drive critical physiological angiogenesis by activating the CYP4A11-20-HETE signaling axis via a HIF-1α-dependent mechanism. The findings from this study potentially identify novel therapeutic targets for the treatment of ischemia and other diseases associated with abnormal angiogenesis.
Subject(s)
Hypochlorous Acid , Peroxidase , Animals , Endothelial Cells/metabolism , Hydroxyeicosatetraenoic Acids , Hypochlorous Acid/metabolism , Hypochlorous Acid/pharmacology , Ischemia/metabolism , Mice , Neovascularization, Pathologic/metabolism , Neutrophils/metabolism , Peroxidase/metabolismABSTRACT
20-Hydroxyeicosatetraenoic acid (20-HETE) was recently identified as a novel contributor of ischemia-induced neovascularization based on the key observation that pharmacological interferences of CYP4A/20-HETE decrease ischemic neovascularization. The objective of the present study is to examine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPCs) and preexisting endothelial cells (ECs). We found that ischemia leads to a time-dependent increase of cyp4a12 expression and 20-HETE production, which are endothelial in origin, using immunofluorescent microscopy, Western blot analysis, and LC-MS/MS. This is accompanied by increases in the tissue stromal cell-derived factor-1α (SDF-1α) expressions as well as SDF-1α plasma levels, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of CYP4A/20-HETE with a 20-HETE synthesis inhibitor, dibromo-dodecenyl-methylsulfimide (DDMS), or a 20-HETE antagonist, N-(20-hydroxyeicosa-6(Z), 15(Z)-dienoyl) glycine (6, 15-20-HEDGE), significantly attenuated these increases. Importantly, we also determined that 20-HETE plays a novel role in maintaining EPC functions and increasing the expression of Oct4, Sox2, and Nanog, which are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of CYP4A/20-HETE decrease the EPC population in culture, whereas 20-HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM-1 expression in the preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15-20-HEDGE. Taken together, CYP4A/20-HETE regulates ischemia-induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization. NEW & NOTEWORTHY CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) was recently discovered as a novel contributor of ischemia-induced neovascularization. However, the underlying molecular and cellular mechanisms are completely unknown. Here, we show that CYP4A/20-HETE regulates the ischemic neovascularization process via its combined actions on both endothelial progenitor cells (EPCs) and preexisting endothelial cells. Moreover, this is the first study, to the best of our knowledge, that associates CYP4A/20-HETE with EPC differentiation and stemness.
Subject(s)
Cytochrome P-450 CYP4A/metabolism , Endothelial Cells/enzymology , Endothelial Progenitor Cells/enzymology , Hydroxyeicosatetraenoic Acids/metabolism , Ischemia/enzymology , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Animals , Cells, Cultured , Chemokine CXCL12/metabolism , Cytochrome P450 Family 4/metabolism , Disease Models, Animal , Hindlimb , Humans , Ischemia/physiopathology , Male , Mice, Inbred BALB C , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Signal Transduction , Time FactorsABSTRACT
Angiogenesis is an important adaptation for recovery from peripheral ischemia. Here, we determined whether 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia-induced angiogenesis and assessed its underlying molecular and cellular mechanisms using a mouse hindlimb-ischemia angiogenesis model. Hindlimb blood flow was measured by Laser Doppler Perfusion Imaging and microvessel density was determined by CD31 and tomato lectin staining. We found that systemic and local administration of a 20-HETE synthesis inhibitor, DDMS, or a 20-HETE antagonist, 6,15-20-HEDGE significantly reduced blood flow recovery and microvessel formation in response to ischemia. 20-HETE production, measured by LC/MS/MS, was markedly increased in ischemic muscles (91±11 vs. 8±2pg/mg in controls), which was associated with prominent upregulation of the 20-HETE synthase, CYP4A12. Immunofluorescence co-localized increased CYP4A12 expression in response to ischemia to CD31-positive EC in the ischemic hindlimb microvessels. We further showed that ischemia increased HIF-1α, VEGF, and VEGFR2 expression in gracilis muscles and that these increases were negated by DDMS and 6,15-20-HEDGE. Lastly, we showed that ERK1/2 of MAPK is a component of 20-HETE regulated ischemic angiogenesis. Taken together, these data indicate that 20-HETE is a critical contributor of ischemia-induced angiogenesis in vivo.
Subject(s)
Hydroxyeicosatetraenoic Acids/metabolism , Ischemia/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Signal Transduction , Angiogenesis Inducing Agents/pharmacology , Angiogenesis Inhibitors/pharmacology , Animals , Blood Flow Velocity , Cell Hypoxia , Cells, Cultured , Cytochrome P450 Family 4/metabolism , Endothelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Hindlimb , Humans , Hydroxyeicosatetraenoic Acids/antagonists & inhibitors , Hydroxyeicosatetraenoic Acids/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ischemia/drug therapy , Ischemia/physiopathology , Mice, Inbred BALB C , Neovascularization, Physiologic/drug effects , Regional Blood Flow , Signal Transduction/drug effects , Time Factors , Up-Regulation , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
RATIONALE: Vascular tissues produce carbon monoxide (CO) via HO-dependent and HO-independent mechanisms; the former in tandem with biliverdin and iron and the latter as a lone product. CO has been shown to function as both a vasoconstrictor and vasodilator; however, factors that dictate the vasoregulatory phenotype of this gas are unknown. OBJECTIVE: We investigated whether CO-mediated vasoconstriction is mechanistically linked to enhanced reactive oxygen species production that masks vasodilatory pathways. METHODS AND RESULTS: Sprague-Dawley rat interlobar and interlobular arteries were examined in terms of superoxide (O2*-) generation and vascular reactivity in the absence and presence of antioxidants. Both authentic CO and the CO-releasing molecule (CORM)-3 constricted renal arteries and increased O2*- production in a dose-dependent manner. The antioxidants tempol, ebselen, and deferoxamine inhibited CO-induced O2*- production and converted CO from constrictor to dilator. CO-induced O2*- generation was found to involve the activity of multiple oxidases including nitric oxide synthase, NADPH oxidase, xanthine oxidase, and complex IV of the mitochondrial electron chain. Furthermore, inhibition of these enzymes converted CO from constrictor to dilator. Similarly, biliverdin and bilirubin inhibited CO-induced O2*- production and vasoconstriction, allowing for a vasodilatory response to CO to be expressed. CO-induced vasoconstriction was dependent on a non-thromboxane agonist of the thromboxane receptor, whereas vasodilatory mechanisms of CO relied on the activation of soluble guanylate cyclase and calcium-gated potassium channels. CONCLUSIONS: CO-induced vasoconstriction involves the generation of reactive oxygen species, which, when negated, allows for the expression of vasodilatory pathways which are masked by the primary oxidative stress response to this gas.
