RESUMO
BACKGROUND AND PURPOSE: Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. Here we have sought to identify new, clinically relevant inhibitors of macropinocytosis, using an FDA-approved drug library. EXPERIMENTAL APPROACH: In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC50 values and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis and clathrin-mediated and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to examine the mechanisms by which selected compounds inhibit macropinocytosis. KEY RESULTS: The primary screen identified 14 compounds that at ~10 µM concentration inhibit >95% of macropinocytotic solute internalization. Three compounds - imipramine, phenoxybenzamine and vinblastine - potently inhibited (IC50 ≤ 131 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Scanning electron microscopy imaging indicated that imipramine inhibits membrane ruffle formation, a critical early step leading to initiation of macropinocytosis. Finally, imipramine has been shown to inhibit macropinocytosis in several cell types, including cancer cells, dendritic cells and macrophages. CONCLUSIONS AND IMPLICATIONS: Our results identify imipramine as a new pharmacological tool to study macropinocytosis in cellular and biological systems. This study also suggests that imipramine could be a good candidate for repurposing as a therapeutic agent in pathological processes involving macropinocytosis.
Assuntos
Aprovação de Drogas/legislação & jurisprudência , Pinocitose/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Clatrina/metabolismo , Células Dendríticas/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Endocitose , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Imipramina/farmacologia , Concentração Inibidora 50 , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Autoantibodies to the ANG II type I receptor (AT1-AA) are associated with preeclampsia (PE). We found that vitamin D supplementation reduced AT1-AA and blood pressure (MAP) in the RUPP rat model of PE. However, it was undetermined whether the decrease in AT1-AA was the mechanism whereby vitamin D lowered MAP or if it were through factors downstream of AT1-AA. Uterine artery resistance index, placental ET-1, and soluble FMS-like tyrosine kinase-1 are increased with AT1-AA-induced hypertension and are considered markers of PE in pregnant women. Therefore, we hypothesized that vitamin D would reduce PE factors during AT1-AA-induced hypertension and could lower blood pressure in a model of hypertension during pregnancy without PE features. Either ANG II (50 ng·kg-1·day) or AT1-AA (1:40) was infused from gestational day (GD) 12-19. vitamin D2 (VD2, 270 IU/day) or vitamin D3 (VD3, 15 IU/day) was administered orally from GD14-GD18. MAP (mmHg) increased in AT1-AA (121 ± 4) and ANG II (113 ± 1)-infused pregnant rats compared with normal pregnant rats (NP) (101 ± 2) but was lower in AT1-AA+VD2 (105 ± 2), AT1-AA+VD3 (109 ± 2), ANG II+VD2 (104 ± 4), and ANG II+VD3 (104 ± 3). VD2 and/or VD3 improved PE features associated with AT1-AA during pregnancy, while ANG II did not induce such features, supporting the hypothesis that AT1-AA induces PE features during pregnancy, and these are improved with vitamin D. In this study, we demonstrate that vitamin D improved many factors associated with PE and reduced blood pressure in a hypertensive model without PE features, indicating that vitamin D could be beneficial for various hypertensive disorders of pregnancy.
Assuntos
Pressão Sanguínea/imunologia , Suplementos Nutricionais , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/fisiopatologia , Receptor Tipo 1 de Angiotensina/imunologia , Vitamina D/administração & dosagem , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Gravidez , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
Deficiency of vitamin D (VD) is associated with preeclampsia (PE), a hypertensive disorder of pregnancy characterized by proinflammatory immune activation. We sought to determine whether VD supplementation would reduce the pathophysiology and hypertension associated with the reduced uterine perfusion pressure (RUPP) rat model of PE. Normal pregnant (NP) and RUPP rats were supplemented with VD2 or VD3 (270 IU and 15 IU/day, respectively) on gestation days 14-18 and mean arterial pressures (MAPs) measured on day 19. MAP increased in RUPP to 123 ± 2 mmHg compared with 102 ± 3 mmHg in NP and decreased to 113 ± 3 mmHg with VD2 and 115 ± 3 mmHg with VD3 in RUPP rats. Circulating CD4+ T cells increased in RUPP to 7.90 ± 1.36% lymphocytes compared with 2.04 ± 0.67% in NP but was lowered to 0.90 ± 0.19% with VD2 and 4.26 ± 1.55% with VD3 in RUPP rats. AT1-AA, measured by chronotropic assay, decreased from 19.5 ± 0.4 bpm in RUPPs to 8.3 ± 0.5 bpm with VD2 and to 15.4 ± 0.7 bpm with VD3. Renal cortex endothelin-1 (ET-1) expression was increased in RUPP rats (11.6 ± 2.1-fold change from NP) and decreased with both VD2 (3.3 ± 1.1-fold) and VD3 (3.1 ± 0.6-fold) supplementation in RUPP rats. Plasma-soluble FMS-like tyrosine kinase-1 (sFlt-1) was also reduced to 74.2 ± 6.6 pg/ml in VD2-treated and 91.0 ± 16.1 pg/ml in VD3-treated RUPP rats compared with 132.7 ± 19.9 pg/ml in RUPP rats. VD treatment reduced CD4+ T cells, AT1-AA, ET-1, sFlt-1, and blood pressure in the RUPP rat model of PE and could be an avenue to improve treatment of hypertension in response to placental ischemia.
Assuntos
Suplementos Nutricionais , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/fisiopatologia , Vitamina D/uso terapêutico , Vitaminas/uso terapêutico , Animais , Pressão Arterial/efeitos dos fármacos , Peso ao Nascer/efeitos dos fármacos , Linfócitos T CD4-Positivos , Endotelina-1/biossíntese , Feminino , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Rim/metabolismo , Contagem de Linfócitos , Gravidez , Ratos , Receptor Tipo 1 de Angiotensina/biossíntese , Fluxo Sanguíneo Regional/efeitos dos fármacos , Útero/fisiopatologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Vitamina D/sangue , Vitaminas/sangueRESUMO
We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-ß (transforming growth factor ß) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.
Assuntos
Benzoatos/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Epóxido Hidrolases/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/fisiologia , Ureia/análogos & derivados , Actinas/metabolismo , Albuminúria/prevenção & controle , Animais , Benzoatos/antagonistas & inibidores , Benzoatos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Creatinina/urina , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/metabolismo , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Epóxido Hidrolases/metabolismo , Epóxido Hidrolases/fisiologia , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/fisiologia , Córtex Renal/metabolismo , Masculino , Proteínas de Membrana/urina , Metaloporfirinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ureia/antagonistas & inibidores , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Induction of hemeoxygenase-1 (HO-1) lowers blood pressure and reduces organ damage in hypertensive animal models; however, a potential protective role for HO-1 induction against diabetic-induced glomerular injury remains unclear. We hypothesize that HO-1 induction will protect against diabetes-induced glomerular injury by maintaining glomerular integrity and inhibiting renal apoptosis, inflammation, and oxidative stress. Diabetes was induced with streptozotocin in spontaneously hypertensive rats (SHR) as a model where the coexistence of hypertension and diabetes aggravates the progression of diabetic renal injury. Control and diabetic SHR were randomized to receive vehicle or the HO-1 inducer cobalt protoporphyrin (CoPP). Glomerular albumin permeability was significantly greater in diabetic SHR compared with control, consistent with an increase in apoptosis and decreased glomerular nephrin and α(3)ß(1)-integrin protein expression in diabetic SHR. CoPP significantly reduced albumin permeability and apoptosis and restored nephrin and α(3)ß(1)-integrin protein expression levels in diabetic SHR. Glomerular injury in diabetic SHR was also associated with increases in NF-κB-induced inflammation and oxidative stress relative to vehicle-treated SHR, and CoPP significantly blunted diabetes-induced increases in glomerular inflammation and oxidative stress in diabetic SHR. These effects were specific to exogenous stimulation of HO-1, since incubation with the HO inhibitor stannous mesoporphyrin alone did not alter glomerular inflammatory markers or oxidative stress yet was able to prevent CoPP-mediated decreases in these parameters. These data suggest that induction of HO-1 reduces diabetic induced-glomerular injury and apoptosis and these effects are associated with decreased NF-κB-induced inflammation and oxidative stress.