RESUMO
RATIONALE: Pulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. METHODS: To test this hypothesis, we exposed global (Gpr75-/-) and endothelial cell (EC) GPR75 knockout (EC-Gpr75-/-) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. RESULTS: Chronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75-/- or EC-Gpr75-/- mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75-/-, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. CONCLUSION: These results suggest GPR75 has a significant role in the development of hypoxia-induced PH.
Assuntos
Hipertensão Pulmonar , Humanos , Camundongos , Animais , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Artéria Pulmonar , Ligantes , Células Cultivadas , Camundongos Endogâmicos C57BL , Hipóxia/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Oxigênio/metabolismo , Camundongos KnockoutRESUMO
Vascular function is dynamically regulated and dependent on a bevy of cell types and factors that work in concert across the vasculature. The vasoactive eicosanoid, 20-Hydroxyeicosatetraenoic acid (20-HETE) is a key player in this system influencing the sensitivity of the vasculature to constrictor stimuli, regulating endothelial function, and influencing the renin angiotensin system (RAS), as well as being a driver of vascular remodeling independent of blood pressure elevations. Several of these bioactions are accomplished through the ligand-receptor pairing between 20-HETE and its high-affinity receptor, GPR75. This 20-HETE axis is at the root of various vascular pathologies and processes including ischemia induced angiogenesis, arteriogenesis, septic shock, hypertension, atherosclerosis, myocardial infarction and cardiometabolic diseases including diabetes and insulin resistance. Pharmacologically, several preclinical tools have been developed to disrupt the 20-HETE axis including 20-HETE synthesis inhibitors (DDMS and HET0016), synthetic 20-HETE agonist analogues (20-5,14-HEDE and 20-5,14-HEDGE) and 20-HETE receptor blockers (AAA and 20-SOLA). Systemic or cell-specific therapeutic targeting of the 20-HETE-GPR75 axis continues to be an invaluable approach as studies examine the molecular underpinnings activated by 20-HETE under various physiological settings. In particular, the development and characterization of 20-HETE receptor blockers look to be a promising new class of compounds that can provide a considerable benefit to patients suffering from these cardiovascular pathologies.
Assuntos
Hipertensão , Sistema Renina-Angiotensina , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Hipertensão/metabolismo , Remodelação Vascular , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Aging is associated with a significant deficiency in circulating insulin-like growth factor-1 (IGF-1), which has an important role in the pathogenesis of age-related vascular cognitive impairment (VCI). Impairment of moment-to-moment adjustment of regional cerebral blood flow via neurovascular coupling (NVC) importantly contributes to VCI. Previous studies established a causal link between circulating IGF-1 deficiency and neurovascular dysfunction. Release of vasodilator mediators from activated astrocytes plays a key role in NVC. To determine the impact of impaired IGF-1 signaling on astrocytic function, astrocyte-mediated NVC responses were studied in a novel mouse model of astrocyte-specific knockout of IGF1R (GFAP-CreERT2/Igf1rf/f) and accelerated neurovascular aging. We found that mice with disrupted astrocytic IGF1R signaling exhibit impaired NVC responses, decreased stimulated release of the vasodilator gliotransmitter epoxy-eicosatrienoic acids (EETs), and upregulation of soluble epoxy hydrolase (sEH), which metabolizes and inactivates EETs. Collectively, our findings provide additional evidence that IGF-1 promotes astrocyte health and maintains normal NVC, protecting cognitive health.
Assuntos
Acoplamento Neurovascular , Envelhecimento , Animais , Astrócitos , Encéfalo , Circulação Cerebrovascular , CamundongosRESUMO
[Figure: see text].
Assuntos
Hipertensão , Músculo Liso Vascular , PPAR gama/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Cloreto de Sódio/metabolismo , Resistência Vascular/fisiologia , Vasodilatação/imunologia , Animais , Modelos Animais de Doenças , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Rim/irrigação sanguínea , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Estresse Oxidativo , Transdução de Sinais , Vasodilatação/fisiologiaRESUMO
Whole brain irradiation (WBI, also known as whole brain radiation therapy or WBRT) is a mainstream therapy for patients with identifiable brain metastases and as a prophylaxis for microscopic malignancies. WBI accelerates brain aging, causing progressive cognitive dysfunction in ~ 50% of surviving patients, thus compromising quality of life. The mechanisms responsible for this WBI side effect remain obscure, and there are no effective treatments or prevention strategies. Here, we test the hypothesis that WBI induces astrocyte senescence, which contributes to impaired astrocytic neurovascular coupling (NVC) responses and the genesis of cognitive decline. To achieve this goal, we used transgenic p16-3MR mice, which allows the detection and selective elimination of senescent cells. We subjected these mice to a clinically relevant protocol of fractionated WBI (5 Gy twice weekly for 4 weeks). WBI-treated and control mice were tested for spatial memory performance (radial arm water maze), astrocyte-dependent NVC responses (whisker-stimulation-induced increases in cerebral blood flow, assessed by laser speckle contrast imaging), NVC-related gene expression, astrocytic release of eicosanoid gliotransmitters and the presence of senescent astrocytes (by flow cytometry, immunohistochemistry and gene expression profiling) at 6 months post-irradiation. WBI induced senescence in astrocytes, which associated with NVC dysfunction and impaired performance on cognitive tasks. To establish a causal relationship between WBI-induced senescence and NVC dysfunction, senescent cells were depleted from WBI-treated animals (at 3 months post-WBI) by genetic (ganciclovir treatment) or pharmacological (treatment with the BCL-2/BCL-xL inhibitor ABT263/Navitoclax, a known senolytic drug) means. In WBI-treated mice, both treatments effectively eliminated senescent astrocytes, rescued NVC responses, and improved cognitive performance. Our findings suggest that the use of senolytic drugs can be a promising strategy for preventing the cognitive impairment associated with WBI.
Assuntos
Astrócitos , Cognição , Acoplamento Neurovascular , Preparações Farmacêuticas , Animais , Encéfalo/efeitos da radiação , Senescência Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Qualidade de Vida , Lesões por RadiaçãoRESUMO
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.
Assuntos
Citocromo P-450 CYP4A/metabolismo , Células Endoteliais/enzimologia , Células Progenitoras Endoteliais/enzimologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Animais , Células Cultivadas , Quimiocina CXCL12/metabolismo , Família 4 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Membro Posterior , Humanos , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos BALB C , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Our aim is to examine the role of PGF2α receptor (FP), a highly expressed prostaglandin receptor in the distal convoluted tubule (DCT) in regulating the basolateral 40-pS K channel. The single-channel studies demonstrated that PGF2α had a biphasic effect on the 40-pS K channel in the DCT-PGF2α stimulated at low concentrations (less than 500 nM), while at high concentrations (above 1 µM), it inhibited the 40-pS K channels. Moreover, neither 13,14-dihydro-15-keto-PGF2α (a metabolite of PGF2α) nor PGE2 was able to mimic the effect of PGF2α on the 40-pS K channel in the DCT. The inhibition of PKC had no significant effect on the 40-pS K channel; however, it abrogated the inhibitory effect of 5 µM PGF2α on the K channel. Moreover, stimulation of PKC inhibited the 40-pS K channel in the DCT, suggesting that PKC mediates the inhibitory effect of PGF2α on the 40-pS K channel. Conversely, the stimulatory effect of PGF2α on the 40-pS K channel was absent in the DCT treated with DPI, a NADPH oxidase (NOX) inhibitor. Also, adding 100 µM H2O2 mimicked the stimulatory effect of PGF2α and increased the 40-pS K channel activity in DCT. Moreover, the stimulatory effect of 500 nM PGF2α and H2O2 was not additive, suggesting the role of superoxide-related species in mediating the stimulatory effect of PGF2α on the 40-pS K channel. The inhibition of Src family tyrosine protein kinase (SFK) not only inhibited the 40-pS K channel in the DCT but also completely abolished the stimulatory effects of PGF2α and H2O2 on the 40-pS K channel. We conclude that PGF2α at low doses stimulates the basolateral 40-pS K channel by a NOX- and SFK-dependent mechanism, while at high concentrations, it inhibits the K channel by a PKC-dependent pathway.
Assuntos
Dinoprosta/farmacologia , Túbulos Renais Distais/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Receptores de Prostaglandina/agonistas , Animais , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Túbulos Renais Distais/metabolismo , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Canais de Potássio/metabolismo , Proteína Quinase C/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
PURPOSE OF REVIEW: 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoactive eicosanoid and a key constituent of the microcirculation. Its effects on vascular function are multifaceted and include stimulation of smooth muscle, contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Such effects have significant implications with regard to the control of vascular homeostasis and pathophysiology. The clinical relevance of 20-HETE is highlighted by recent studies linking 20-HETE and its biosynthetic enzymes to the development of hypertension, stroke, and myocardial infarction. RECENT FINDINGS: This article presents past and recent findings that focus on the role of 20-HETE in the regulation of the vasculature in health and disease and the implication of its actions on endothelial and vascular smooth muscle cells to the pathogenesis of hypertension and stroke. SUMMARY: To date clinical studies corroborated animal studies in that they place 20-HETE as a significant contributor to the pathogenesis of cardiovascular diseases. Consequently, uncovering 20-HETE effects in the vasculature along with understanding its mechanism of action provide a strong basis for the development of novel therapeutic strategies to prevent vascular/end organ damage in these diseases.
Assuntos
Células Endoteliais/fisiologia , Ácidos Hidroxieicosatetraenoicos/fisiologia , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/fisiologia , Animais , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/metabolismoRESUMO
Epoxyeicosatrienoicacids (EETs), synthesized from arachidonic acid by epoxygenases of the CYP2C and CYP2J gene subfamilies, contribute to hypoxic pulmonary vasoconstriction (HPV) in mice. Despite their roles in HPV, it is controversial whether EETs mediate or ameliorate pulmonary hypertension (PH). A recent study showed that deficiency of Cyp2j did not protect male and female mice from hypoxia-induced PH. Since CYP2C44 is a functionally important epoxygenase, we hypothesized that knockout of the Cyp2c44 gene would protect both sexes of mice from hypoxia-induced PH. We tested this hypothesis in wild-type (WT) and Cyp2c44 knockout (Cyp2c44 (-/-)) mice exposed to normoxia (room air) and hypoxia (10% O2) for 5 weeks. Exposure of WT and Cyp2c44 (-/-) mice to hypoxia resulted in pulmonary vascular remodeling, increased pulmonary artery resistance, and decreased cardiac function in both sexes. However, in female Cyp2c44 (-/-) mice, compared with WT mice, (1) pulmonary artery resistance and right ventricular hypertrophy were greater, (2) cardiac index was lower, (3) left ventricular and arterial stiffness were higher, and (4) plasma aldosterone levels were higher, but (5) there was no difference in levels of EET in lungs and heart. Paradoxically and unexpectedly, we found that Cyp2c44 disruption exacerbated hypoxia-induced PH in female but not male mice. We attribute exacerbated PH in female Cyp2c44 (-/-) mice to elevated aldosterone and as-yet-unknown systemic factors. Therefore, we suggest a role for the human CYP2C genes in protecting women from severe PH and that this could be one of the underlying causes for a better 5-year survival rate in women than in men.
RESUMO
Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.
Assuntos
Androgênios/farmacologia , Família 4 do Citocromo P450/genética , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/metabolismo , Guanilil Ciclase Solúvel/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Feminino , Ligação Genética , Hipertensão/genética , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Locos de Características Quantitativas , Fatores Sexuais , Guanilil Ciclase Solúvel/genética , Testosterona/sangueRESUMO
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.
Assuntos
Ácidos Hidroxieicosatetraenoicos/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Transdução de Sinais , Indutores da Angiogênese/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Velocidade do Fluxo Sanguíneo , Hipóxia Celular , Células Cultivadas , Família 4 do Citocromo P450/metabolismo , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Membro Posterior , Humanos , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/tratamento farmacológico , Isquemia/fisiopatologia , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18ß-glycyrrhetinic acid (18ß-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18ß-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18ß-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18ß-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.
Assuntos
Junções Comunicantes/fisiologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Miócitos de Músculo Liso/fisiologia , Vasoconstrição , Animais , Bovinos , Hipóxia Celular , Células Cultivadas , Células Endoteliais , Junções Comunicantes/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Artéria Pulmonar/citologiaRESUMO
20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inflamação/prevenção & controle , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Bovinos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citocromo P-450 CYP4A/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genótipo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Inflamação/enzimologia , Inflamação/genética , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Mutantes , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias Musculares/enzimologia , Músculo Liso Vascular/enzimologia , Mutação , Miócitos de Músculo Liso/enzimologia , Fenótipo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Increased CYP epoxygenase activity and consequently up regulation of epoxyeicosatrienoic acids (EETs) levels provides protection against metabolic syndrome and cardiovascular diseases. Conversion of arachidonic acid epoxides to diols by soluble epoxide hydrolase (sEH) diminishes the beneficial cardiovascular properties of these epoxyeicosanoids. We therefore examined the possible biochemical consequences of sEH deletion on vascular responses in male and female mice. Through the use of the sEH KO mouse, we provide evidence of differences in the compensatory response in the balance between nitric oxide (NO), carbon monoxide (CO), EETs and the vasoconstrictor 20-HETE in male and female KO mice. Serum levels of adiponectin, TNFα, IL-1b and MCP1 and protein expression in vascular tissue of p-AMPK, p-AKT and p-eNOS were measured. Deletion of sEH caused a significant (p<0.05) decrease in body weight, and an increase in adiponectin, pAMPK and pAKT levels in female KO mice compared to male KO mice. Gene deletion resulted in a higher production of renal EETs in female KO compared to male KO mice and, concomitantly, we observed an increase in renal 20-HETEs levels and superoxide anion production only in male KO mice. sEH deletion increased p-AKT and p-eNOS protein expression but decreased p-AMPK levels in female KO mice. Increased levels of p-eNOS at Thr-495 were observed only in KO male mice. While p-eNOS at 1177 were not significantly different between male and female. Nitric oxide production was unaltered in male KO mice. These results provide evidence of gender differences in the preservation of vascular homeostasis in response to sEH deletion which involves regulation of phosphorylation of eNOS at the 495 site.
Assuntos
Vasos Sanguíneos/metabolismo , Epóxido Hidrolases/deficiência , Epóxido Hidrolases/genética , Técnicas de Inativação de Genes , Homeostase/genética , Caracteres Sexuais , Animais , Peso Corporal/genética , Citocinas/sangue , Eicosanoides/metabolismo , Epóxido Hidrolases/química , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Ácidos Hidroxieicosatetraenoicos/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Solubilidade , Superóxidos/metabolismo , Treonina/metabolismoRESUMO
20-hydroxyeicosatetraenoic acid (20-HETE) is a metabolite of arachidonic acid that exhibits a myriad of biological effects in the vascular system. This review discusses the current knowledge related to the effects of 20-HETE on vascular reactivity, activation, and remodeling, as well as its role in vascular inflammation and angiogenesis. The information explaining how 20-HETE and the renin-angiotensin system interact to promote hypertension, vasoconstriction, and vascular dysfunction is summarized in this article. 20-HETE enhances vascular inflammation and injury in models of diabetes, ischemia/reperfusion, and cerebrovascular oxidative stress. Recent studies also established a role for 20-HETE in normal and pathological angiogenesis conditions. This review will also discuss the molecular mechanisms through which 20-HETE induces these vascular actions. Potential additional studies are suggested to address shortcomings in the current knowledge of 20-HETE in the vascular system.
Assuntos
Vasos Sanguíneos/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Animais , Vasos Sanguíneos/patologia , Vasos Sanguíneos/fisiologia , Humanos , Neovascularização Fisiológica , Sistema Renina-Angiotensina , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Remodelação VascularRESUMO
20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin-angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/-). Treatment of male AGT+/- with 5α-dihydrotestosterone (DHT) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. DHT increased vascular 20-HETE levels in AGT+/- and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/- and WT mice. Administration of 20-HEDE at the peak of the DHT-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/- compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/- with DHT decreased the levels of EETs (28.4±5.1ng/mg). DHT-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/- and WT by 30-40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing DHT-mediated increases in BP in both AGT+/- and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.
Assuntos
Androgênios/efeitos adversos , Angiotensinas/deficiência , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Hipertensão , Androgênios/farmacologia , Animais , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/genética , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Hipertensão/genética , Hipertensão/metabolismo , Masculino , Camundongos , Camundongos MutantesRESUMO
Heme oxygenase (HO)-2 deficiency impairs wound healing and exacerbates inflammation following injury. We examine the impact of HO-2 deficiency on macrophage function and the contribution of macrophage HO-2 to inflammatory and repair responses to injury. Corneal epithelial debridement was performed in control and macrophage-depleted HO-2(-/-) and wild-type (WT) mice and in bone marrow chimeras. Peritoneal macrophages were collected for determination of phagocytic activity and classically activated macrophage (M1)-alternatively activated macrophage (M2) polarization. Depletion of macrophages delayed corneal healing (13.2%) and increased neutrophil infiltration (54.1%) by day 4 in WT mice, whereas in HO-2(-/-) mice, it did not worsen the already impaired wound healing and exacerbated inflammation. HO-2(-/-) macrophages displayed an altered M1 phenotype with no significant expression of M2 or M2-like activated cells and a 31.3% reduction in phagocytic capacity that was restored by inducing HO-1 activity or supplementing biliverdin. Macrophage depletion had no effect, whereas adoptive transfer of WT bone marrow improved wound healing (34% on day 4) but did not resolve the exaggerated inflammatory response in HO-2(-/-) mice. These findings indicate that HO-2-deficient macrophages are dysfunctional and that macrophage HO-2 is required for proper macrophage function but is insufficient to correct the impaired healing of the HO-2(-/-) cornea, suggesting that corneal epithelial expression of HO-2 is a key to resolution and repair in wound healing.
Assuntos
Lesões da Córnea/fisiopatologia , Heme Oxigenase (Desciclizante)/deficiência , Macrófagos/enzimologia , Macrófagos/fisiologia , Cicatrização/fisiologia , Animais , Transplante de Medula Óssea , Lesões da Córnea/patologia , Citocinas/biossíntese , Epitélio Corneano/patologia , Epitélio Corneano/fisiopatologia , Feminino , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Ativação de Macrófagos , Macrófagos/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/fisiologia , Fagocitose , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Quimeras de Transplante/fisiologiaRESUMO
In the kidney, 20-hydroxyeicosatetraenoic acid (20-HETE) is a primary cytochrome P450 4 (Cyp4)-derived eicosanoid that enhances vasoconstriction of renal vessels and induces hypertension, renal tubular cell hypertrophy, and podocyte apoptosis. Hypertension and podocyte injury contribute to diabetic nephropathy and are strong predictors of disease progression. In this study, we defined the mechanisms whereby 20-HETE affects the progression of diabetic nephropathy. We used Cyp4a14KO male mice that exhibit androgen-sensitive hypertension due to increased Cyp4a12-mediated 20-HETE production. We show that, upon induction of diabetes type 1 via streptozotocin injection, Cyp4a14KO male mice developed worse renal disease than streptozotocin-treated wild-type mice, characterized by increased albuminuria, mesangial expansion, glomerular matrix deposition, and thickness of the glomerular basement membranes. Castration blunted androgen-mediated Cyp4a12 synthesis and 20-HETE production, normalized BP, and ameliorated renal damage in diabetic Cyp4a14KO mice. Notably, treatment with a 20-HETE antagonist or agents that normalized BP without affecting Cyp4a12 expression and 20-HETE biosynthesis also ameliorated diabetes-mediated renal damage and albuminuria in Cyp4a14KO male mice. Taken together, these results suggest that hypertension is the major contributor to 20-HETE-driven diabetes-mediated kidney injury.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Nefropatias Diabéticas/etiologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/complicações , Animais , Colágeno/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Família 4 do Citocromo P450 , Nefropatias Diabéticas/patologia , Membrana Basal Glomerular/patologia , Hidralazina , Hidroclorotiazida , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Masculino , Camundongos Knockout , Orquiectomia , Sistema Renina-Angiotensina , Reserpina , Sódio/metabolismoRESUMO
Obesity is a global epidemic and a common risk factor for endothelial dysfunction and the subsequent development of diabetes mellitus and vascular diseases such as hypertension. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-derived metabolites of arachidonic acid that contribute to vascular protection by stimulating vasodilation and inhibiting inflammation. Heme oxygenase-1 is a stress response protein that plays an important cytoprotective role against oxidative insult in diabetes mellitus and cardiovascular disease. We recently demonstrated interplay between EETs and heme oxygenase-1 in the attenuation of adipogenesis. We examined whether adipocyte dysfunction in mice fed a high-fat diet could be prevented by endothelial-specific targeting of the human CYP epoxygenase, CYP2J2. Tie2-CYP2J2 transgenic mice, fed a high-fat diet, had a reduction in body weight gain, blood glucose, insulin levels, and inflammatory markers. Tie2-CYP2J2 gene targeting restored HF-mediated decreases in vascular heme oxygenase-1, Cyp2C44, soluble epoxide hydrolase, phosphorylated endothelial nitric oxide synthase, phosphorylated protein kinase B, and phosphorylated adenosine monophosphate protein kinase protein expression, thus improving vascular function. These changes translated into decreased inflammation and oxidative stress within adipose tissue and decreased peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein alpha, mesoderm-specific transcript, and adipocyte 2 expression and increased uncoupling protein 1 and uncoupling protein 2 expression, reflecting the effect of vascular EET overproduction on adipogenesis. The current study documents a direct link between endothelial-specific EET production and adipogenesis, further implicating the EET-heme oxygenase-1 crosstalk as an important cytoprotective mechanism in the amelioration of vascular and adipocyte dysfunction resulting from diet-induced obesity.
Assuntos
Adiposidade/genética , Sistema Enzimático do Citocromo P-450/genética , DNA/genética , Regulação da Expressão Gênica , Obesidade/genética , Doenças Vasculares/genética , Vasodilatação/fisiologia , Adipócitos/metabolismo , Adipócitos/patologia , Ração Animal , Animais , Western Blotting , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/biossíntese , Gorduras na Dieta/farmacologia , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Obesidade/complicações , Obesidade/metabolismo , Fenótipo , Pletismografia , Doenças Vasculares/etiologia , Doenças Vasculares/fisiopatologiaRESUMO
Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the role of CYP4F2 in vascular disease, we generated mice with endothelial expression of human CYP4F2 (Tie2-CYP4F2-Tr). LC/MS/MS analysis revealed 2-foldincreases in 20-HETE levels in tissues and endothelial cells (ECs), relative to wild-type (WT) controls. Tie2-CYP4F2-Tr ECs demonstrated increases in growth (267.1 ± 33.4 vs. 205.0 ± 13% at 48 h) and tube formation (7.7 ± 1.1 vs. 1.6 ± 0.5 tubes/field) that were 20-HETE dependent and associated with up-regulation of prooxidant NADPH oxidase and proangiogenic VEGF. Increases in VEGF and NADPH oxidase levels were abrogated by inhibitors of NADPH oxidase and MAPK, respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-CYP4F2-Tr mice (18.6 ± 2.7 vs. 7.9 ± 2.7 pg/ml) were up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and blood pressure were unchanged. Our findings indicate that human CYP4F2 significantly increases 20-HETE production, CYP4F2-derived 20-HETE mediates EC proliferation and angiogenesis via VEGF- and NADPH oxidase-dependent manners, and the Tie2-CYP4F2-Tr mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the vasculature.-Cheng, J., Edin, M. L., Hoopes, S. L., Li, H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih, F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake, G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.