RESUMO
Aging is a major risk factor for cerebral infarction. Since cellular senescence is intrinsic to aging, we postulated that stroke-induced cellular senescence might contribute to neural dysfunction. Adult male Wistar rats underwent 60-minute middle cerebral artery occlusion and were grouped according to 3 reperfusion times: 24 hours, 3, and 7 days. The major biomarkers of senescence: 1) accumulation of the lysosomal pigment, lipofuscin; 2) expression of the cell cycle arrest markers p21, p53, and p16INK4a; and 3) expression of the senescence-associated secretory phenotype cytokines interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin-1ß (IL-1ß) were investigated in brain samples. Lipofuscin accumulation was scarce at the initial stage of brain damage (24 hours), but progressively increased until it reached massive distribution at 7 days post-ischemia. Lipofuscin granules (aggresomes) were mainly confined to the infarcted areas, that is parietal cortex and adjacent caudate-putamen, which were equally affected. The expression of p21, p53, and p16INK4a, and that of IL-6, TNF-α, and IL-1ß, was significantly higher in the ischemic hemisphere than in the non-ischemic hemisphere. These data indicate that brain cell senescence develops during acute ischemic infarction and suggest that the acute treatment of ischemic stroke might be enhanced using senolytic drugs.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Senescência Celular , Infarto da Artéria Cerebral Média/metabolismo , Interleucina-6 , Lipofuscina/metabolismo , Masculino , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa , Proteína Supressora de Tumor p53/metabolismoRESUMO
Background: Rehabilitation is still the only treatment available to improve functional status after the acute phase of stroke. Most clinical guidelines highlight the need to design rehabilitation treatments considering starting time, intensity, and frequency, according to the tolerance of the patient. However, there are no homogeneous protocols and the biological effects are under investigation. Objective: To investigate the impact of rehabilitation intensity (hours) after stroke on functional improvement and serum angiogenin (ANG) in a 6-month follow-up study. Methods: A prospective, observational, longitudinal, and multicenter study with three cohorts: strokes in intensive rehabilitation therapy (IRT, minimum 15 h/week) vs. conventional therapy (NO-IRT, <15 h/week), and controls subjects (without known neurological, malignant, or inflammatory diseases). A total of seven centers participated, with functional evaluations and blood sampling during follow-up. The final cohort includes 62 strokes and 43 controls with demographic, clinical, blood samples, and exhaustive functional monitoring. Results: The median (IQR) number of weekly hours of therapy was different: IRT 15 (15-16) vs. NO-IRT 7.5 (5-9), p < 0.01, with progressive and significant improvements in both groups. However, IRT patients showed earlier improvements (within 1 month) on several scales (CAHAI, FMA, and FAC; p < 0.001) and the earliest community ambulation achievements (0.89 m/s at 3 months). There was a significant difference in ANG temporal profile between the IRT and NO-IRT groups (p < 0.01). Additionally, ANG was elevated at 1 month only in the IRT group (p < 0.05) whereas it decreased in the NO-IRT group (p < 0.05). Conclusions: Our results suggest an association of rehabilitation intensity with early functional improvements, and connect the rehabilitation process with blood biomarkers.
RESUMO
Despite the promising neuroprotective effects of uric acid (UA) in acute ischemic stroke, the seemingly pleiotropic underlying mechanisms are not completely understood. Recent evidence points to transcription factors as UA targets. To gain insight into the UA mechanism of action, we investigated its effects on pertinent biomarkers for the most relevant features of ischemic stroke pathophysiology: (1) oxidative stress (antioxidant enzyme mRNAs and MDA), (2) neuroinflammation (cytokine and Socs3 mRNAs, STAT3, NF-κB p65, and reactive microglia), (3) brain swelling (Vegfa, Mmp9, and Timp1 mRNAs), and (4) apoptotic cell death (Bcl-2, Bax, caspase-3, and TUNEL-positive cells). Adult male Wistar rats underwent intraluminal filament transient middle cerebral artery occlusion (tMCAO) and received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at 20 min reperfusion. The outcome measures were neurofunctional deficit, infarct, and edema. UA treatment reduced cortical infarct and brain edema, as well as neurofunctional impairment. In brain cortex, increased UA: (1) reduced tMCAO-induced increases in Vegfa and Mmp9/Timp1 ratio expressions; (2) induced Sod2 and Cat expressions and reduced MDA levels; (3) induced Il6 expression, upregulated STAT3 and NF-κB p65 phosphorylation, induced Socs3 expression, and inhibited microglia activation; and (4) ameliorated the Bax/Bcl-2 ratio and induced a reduction in caspase-3 cleavage as well as in TUNEL-positive cell counts. In conclusion, the mechanism for morphological and functional neuroprotection by UA in ischemic stroke is multifaceted, since it is associated to activation of the IL-6/STAT3 pathway, attenuation of edematogenic VEGF-A/MMP-9 signaling, and modulation of relevant mediators of oxidative stress, neuroinflammation, and apoptotic cell death.
Assuntos
Interleucina-6/metabolismo , AVC Isquêmico/metabolismo , Neuroproteção/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Ácido Úrico/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Edema Encefálico/etiologia , Edema Encefálico/patologia , Edema Encefálico/fisiopatologia , Infarto Encefálico/etiologia , Infarto Encefálico/fisiopatologia , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/fisiopatologia , AVC Isquêmico/etiologia , AVC Isquêmico/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ácido Úrico/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Progression on therapy in non-small cell lung carcinoma (NSCLC) is often evaluated radiographically, however, image-based evaluation of said therapies may not distinguish disease progression due to intrinsic tumor drug resistance or inefficient tumor penetration of the drugs. Here we report that the inhibition of mutated EGFR promotes the secretion of a potent vasoconstrictor, endothelin-1 (EDN1), which continues to increase as the cells become resistant with a mesenchymal phenotype. As EDN1 and its receptor (EDNR) is linked to cancer progression, EDNR-antagonists have been evaluated in several clinical trials with disappointing results. These trials were based on a hypothesis that the EDN1-EDNR axis activates the MAPK-ERK signaling pathway that is vital to the cancer cell survival; the trials were not designed to evaluate the impact of tumor-derived EDN1 in modifying tumor microenvironment or contributing to drug resistance. Ectopic overexpression of EDN1 in cells with mutated EGFR resulted in poor drug delivery and retarded growth in vivo but not in vitro. Intratumoral injection of recombinant EDN significantly reduced blood flow and subsequent gefitinib accumulation in xenografted EGFR-mutant tumors. Furthermore, depletion of EDN1 or the use of endothelin receptor inhibitors bosentan and ambrisentan improved drug penetration into tumors and restored blood flow in tumor-associated vasculature. Correlatively, these results describe a simplistic endogenous yet previously unrealized resistance mechanism inherent to a subset of EGFR-mutant NSCLC to attenuate tyrosine kinase inhibitor delivery to the tumors by limiting drug-carrying blood flow and the drug concentration in tumors. SIGNIFICANCE: EDNR antagonists can be repurposed to improve drug delivery in VEGFA-secreting tumors, which normally respond to TKI treatment by secreting EDN1, promoting vasoconstriction, and limiting blood and drug delivery.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Endotelina-1/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Disponibilidade Biológica , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endotelina-1/genética , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacocinética , Humanos , Neoplasias Pulmonares/genética , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because neuroprotection in stroke should be revisited in the era of recanalisation, the present study analysed the potential neuroprotective effect of the selective oestrogen receptor modulator, bazedoxifene acetate (BZA), in an animal model of diabetic ischaemic stroke that mimics thrombectomy combined with adjuvant administration of a putative neuroprotectant. Four weeks after induction of diabetes (40 mg kg-1 streptozotocin, i.p.), male Wistar rats were subjected to transient middle cerebral artery occlusion (intraluminal thread technique, 60 minutes) and assigned to one of three groups treated with either: vehicle, BZA (3 mg kg-1 day-1 , i.p.) or 17ß-oestradiol (E2 ) (100 µg kg-1 day-1 , i.p.). At 24 hours post-ischaemia-reperfusion, brain damage (neurofunctional score, infarct size and apoptosis), expression of oestrogen receptors (ER)α, ERß and G protein-coupled oestrogen receptor), and activity of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)1/2 and phosphoinositide 3-kinase/Akt pathways were analysed. At 24 hours after the ischaemic insult, both BZA- and E2 -treated animals showed lower brain damage in terms of improved neurofunctional condition, decreased infarct size and decreased apoptotic cell death. Ischaemia-reperfusion induced a significant decrease in ERα and ERß expression without affecting that of G protein-coupled oestrogen receptor, whereas BZA and E2 reversed such a decrease. The ischaemic insult up-regulated the activity of both the MAPK/ERK1/2 and phosphoinositide 3-kinase/Akt pathways; BZA and E2 attenuated the increased activity of the ERK1/2 pathway, without affecting that of the Akt pathway. The results of the present study lend further support to the consideration of BZA as an effective and safer alternative overcoming the drawbacks of E2 with respect to improving diabetic ischaemic stroke outcome after successful reperfusion.
Assuntos
Isquemia Encefálica/prevenção & controle , Angiopatias Diabéticas/prevenção & controle , Estradiol/farmacologia , Indóis/farmacologia , Receptores de Estrogênio/genética , Acidente Vascular Cerebral/prevenção & controle , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Estreptozocina , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
Along with its role in regulating blood pressure and fluid homeostasis, the natriuretic peptide system could be also part of an endogenous protective mechanism against brain damage. We aimed to assess the possibility that exogenous atrial natriuretic peptide (ANP) could protect against acute ischemic stroke, as well as the molecular mechanisms involved. Three groups of rats subjected to transient middle cerebral artery occlusion (tMCAO, intraluminal filament technique, 60â¯min) received intracerebroventricular vehicle, low-dose ANP (0.5â¯nmol) or high-dose ANP (2.5â¯nmol), at 30â¯min reperfusion. Neurofunctional condition, and brain infarct and edema volumes were measured at 24â¯h after tMCAO. Apoptotic cell death and expression of natriuretic peptide receptors (NPR-A and NPR-C), K+ channels (KATP, KV and BKCa), and PI3K/Akt and MAPK/ERK1/2 signaling pathways were analyzed. Significant improvement in neurofunctional status, associated to reduction in infarct and edema volumes, was shown in the high-dose ANP group. As to the molecular mechanisms analyzed, high-dose ANP: 1) reduced caspase-3-mediated apoptosis; 2) did not modify the expression of NPR-A and NPR-C, which had been downregulated by the ischemic insult; 3) induced a significant reversion of ischemia-downregulated KATP channel expression; and 4) induced a significant reversion of ischemia-upregulated pERK2/ERK2 expression ratio. In conclusion, ANP exerts a significant protective role in terms of both improvement of neurofunctional status and reduction in infarct volume. Modulation of ANP on some molecular mechanisms involved in ischemia-induced apoptotic cell death (KATP channels and MAPK/ERK1/2 signaling pathway) could account, at least in part, for its beneficial effect. Therefore, ANP should be considered as a potential adjunctive neuroprotective agent improving stroke outcome after successful reperfusion interventions.
Assuntos
Fator Natriurético Atrial/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Fator Natriurético Atrial/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Isquemia Encefálica/complicações , Caspase 3/metabolismo , Clivagem do DNA/efeitos dos fármacos , Regulação para Baixo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Injeções Intraventriculares , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Potássio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Receptores do Fator Natriurético Atrial/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/complicaçõesRESUMO
Brain preconditioning (PC) refers to a state of transient tolerance against a lethal insult that can be evoked by a prior mild event. It is thought that PC may induce different pathways responsible for neuroprotection, which may involve the attenuation of cell damage pathways, including the apoptotic cell death. In this context, p53 is a stress sensor that accumulates during brain ischemia leading to neuronal death. The murine double minute 2 gene (MDM2), a p53-specific E3 ubiquitin ligase, is the main cellular antagonist of p53, mediating its degradation by the proteasome. Here, we study the role of MDM2-p53 pathway on PC-induced neuroprotection both in cultured neurons (in vitro) and rat brain (in vivo). Our results show that PC increased neuronal MDM2 protein levels, which prevented ischemia-induced p53 stabilization and neuronal death. Indeed, PC attenuated ischemia-induced activation of the p53/PUMA/caspase-3 signaling pathway. Pharmacological inhibition of MDM2-p53 interaction in neurons abrogated PC-induced neuroprotection against ischemia. Finally, the relevance of the MDM2-p53 pathway was confirmed in rat brain using a PC model in vivo. These findings demonstrate the key role of the MDM2-p53 pathway in PC-induced neuroprotection against a subsequent ischemic insult and poses MDM2 as an essential target in ischemic tolerance.
Assuntos
Encéfalo/patologia , Isquemia/patologia , Precondicionamento Isquêmico , Neurônios/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Camundongos , RatosRESUMO
Despite transferrin being the main circulating carrier of iron in body fluids, and iron overload conditions being known to worsen stroke outcome through reactive oxygen species (ROS)-induced damage, the contribution of blood transferrin saturation (TSAT) to stroke brain damage is unknown. The objective of this study was to obtain evidence on whether TSAT determines the impact of experimental ischemic stroke on brain damage and whether iron-free transferrin (apotransferrin, ATf)-induced reduction of TSAT is neuroprotective. We found that experimental ischemic stroke promoted an early extravasation of circulating iron-loaded transferrin (holotransferrin, HTf) to the ischemic brain parenchyma. In vitro, HTf was found to boost ROS production and to be harmful to primary neuronal cultures exposed to oxygen and glucose deprivation. In stroked rats, whereas increasing TSAT with exogenous HTf was detrimental, administration of exogenous ATf and the subsequent reduction of TSAT was neuroprotective. Mechanistically, ATf did not prevent extravasation of HTf to the brain parenchyma in rats exposed to ischemic stroke. However, ATf in vitro reduced NMDA-induced neuronal uptake of HTf and also both the NMDA-mediated lipid peroxidation derived 4-HNE and the resulting neuronal death without altering Ca2+-calcineurin signaling downstream the NMDA receptor. Removal of transferrin from the culture media or blockade of transferrin receptors reduced neuronal death. Together, our data establish that blood TSAT exerts a critical role in experimental stroke-induced brain damage. In addition, our findings suggest that the protective effect of ATf at the neuronal level resides in preventing NMDA-induced HTf uptake and ROS production, which in turn reduces neuronal damage.
Assuntos
Apoproteínas/administração & dosagem , Isquemia Encefálica/tratamento farmacológico , Sobrecarga de Ferro/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológico , Transferrina/administração & dosagem , Animais , Apoproteínas/sangue , Isquemia Encefálica/sangue , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/patologia , Desferroxamina/administração & dosagem , Feminino , Humanos , Ferro/sangue , Sobrecarga de Ferro/sangue , Sobrecarga de Ferro/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Neurônios/metabolismo , Neurônios/patologia , Ratos , Espécies Reativas de Oxigênio/sangue , Receptores da Transferrina/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia , Transferrina/metabolismoRESUMO
As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERß and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17ß-estradiol (E2)-treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60min), were distributed in vehicle-, BZA- (20.7±2.1ng/mL in plasma) and E2- (45.6±7.8pg/mL in plasma) treated groups. At 24h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERß expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERß expression, E2 increased ERα expression but did not change that of ERß. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERß expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the consideration of BZA as a potential neuroprotective drug in acute ischemic stroke treatment.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Indóis/uso terapêutico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Estradiol/uso terapêutico , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Estrogênios/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fosfatos de Fosfatidilinositol/agonistas , Fosfatos de Fosfatidilinositol/metabolismo , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-ß-estradiol, 4,4',4â³-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4â³-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-ß-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERß agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERß (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).
Assuntos
Artéria Basilar/efeitos dos fármacos , Estrogênios/farmacologia , Indóis/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Artéria Basilar/fisiologia , Relação Dose-Resposta a Droga , Masculino , Técnicas de Cultura de Órgãos , Coelhos , Vasodilatação/fisiologiaRESUMO
Hypertension is one of the most important causes of cardiovascular and renal morbidity and mortality, and it represents a serious health problem in Western countries. Over the last few decades scientific interest in food-derived antihypertensive peptides has grown as an alternative to drugs in the control of systemic blood pressure. Most of these peptides target the angiotensin I-converting enzyme (ACE) but emerging evidence points to other antihypertensive mechanisms beyond ACE inhibition. The milk protein lactoferrin (LF) is a good source of orally active antihypertensive peptides the characterization of which, including ex vivo functional assays and in vivo approaches, shows that they might act on several molecular targets. This review summarizes the mechanisms of action underlying the blood pressure-lowering effects of LF-derived peptides, focusing on their interaction with different components of the renin-angiotensin (RAS) and endothelin (ET) systems. The ability of LF-derived peptides to modify the expression of genes encoding proteins involved in the nitric oxide (NO) pathway and prostaglandin synthesis is also described.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Lactoferrina/farmacologia , Peptídeos/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Anti-Hipertensivos/química , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Lactoferrina/química , Peptídeos/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismoRESUMO
Novel antihypertensive peptides released by Kluyveromyces marxianus from bovine lactoferrin (LF) have been identified. K. marxianus LF permeate was fractionated by semipreparative high performance liquid chromatography and 35 peptides contained in the angiotensin I-converting enzyme (ACE)-inhibitory fractions were identified by using an ion trap mass spectrometer. On the basis of peptide abundance and common structural features, six peptides were chemically synthesized. Four of them (DPYKLRP, PYKLRP, YKLRP, and GILRP) exerted in vitro inhibitory effects on ACE activity and effectively decreased systolic blood pressure after oral administration to spontaneously hypertensive rats (SHRs). Stability against gastrointestinal enzymes suggested that the sequence LRP could contribute to the in vivo effects of parental peptides. Finally, there were reductions in circulating ACE activity and angiotensin II level in SHRs after either DPYKLRP or LRP intake, thus confirming ACE inhibition as the in vivo mechanism for their antihypertensive effect.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Anti-Hipertensivos/administração & dosagem , Hipertensão/tratamento farmacológico , Kluyveromyces/metabolismo , Lactoferrina/metabolismo , Peptídeos/administração & dosagem , Administração Oral , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Biotransformação , Pressão Sanguínea/efeitos dos fármacos , Bovinos , Humanos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Lactoferrina/administração & dosagem , Lactoferrina/química , Masculino , Peptídeos/química , Peptídeos/metabolismo , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Ratos , Ratos Endogâmicos SHRRESUMO
Looking for antihypertensive mechanisms beyond ACE inhibition, we assessed whether lactoferrin (LF)-derived peptides can act as receptor blockers to inhibit vasoconstriction induced by angiotensin II or endothelin-1. The lactoferricin B (LfcinB)-derived peptide LfcinB20-25 (RRWQWR), the low molecular weight LF hydrolysate (LFH < 3 kDa), and two peptides identified in LFH < 3 kDa (LIWKL and RPYL) were tested in ex vivo assays of vasoactive responses. The peptide RPYL was tested in radioligand receptor binding assays. Both LFH < 3 kDa and individual peptides inhibited angiotensin II-induced vasoconstriction. RPYL showed the highest ex vivo inhibitory effect and also inhibited binding of [(125)I]-(Sar(1),Ile(8))-angiotensin II to AT1 receptors. By contrast, neither LFH < 3 kDa nor RPYL inhibited endothelin-1 and depolarization-induced vasoconstrictions. In conclusion, LF-derived peptides selectively inhibit angiotensin II-induced vasoconstriction by blocking angiotensin AT1 receptors. Therefore, inhibition of angiotensin II-induced vasocontriction is suggested as a mechanism contributing along with ACE inhibition to the antihypertensive effect of some LF-derived peptides.
Assuntos
Antagonistas de Receptores de Angiotensina/administração & dosagem , Anti-Hipertensivos/administração & dosagem , Hipertensão/tratamento farmacológico , Lactoferrina/química , Peptídeos/administração & dosagem , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina/química , Animais , Anti-Hipertensivos/química , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Peso Molecular , Peptídeos/química , Coelhos , Receptor Tipo 1 de Angiotensina/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
Bioactive ACE inhibiting peptides are gaining interest in hypertension treatment. We have designed and screened six synthetic heptapeptides (PACEI48 to PACEI53) based on two hexapeptide leads (PACEI32 and PACEI34) to improve ACE inhibitory properties and assess their antihypertensive effects. ACE activity was assayed in vitro and ex vivo. Selected peptides were administered to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. In vitro cytotoxicity was assessed with the MTT reduction test. The six heptapeptides at low micromolar concentration produced different degrees of in vitro inhibition of ACE activity using the synthetic substrate HHL or the natural substrate angiotensin I; and ex vivo inhibition of ACE-dependent, angiotensin I-induced vasoconstriction, but not angiotensin II-induced vasoconstriction. Oral administration of the hexapeptide PACEI32L, and the heptapeptides PACEI50L and PACEI52L, induced reductions in systolic blood pressure lasting up to 3h in SHRs but not in WKY rats. Intravenous injection of PACEI32L and PACEI50L, but not PACEI52L, induced acute transient reductions in mean blood pressure of SHRs. d-Amino acid peptides showed five-fold less ACE inhibitory potency, no inhibitory effect on angiotensin I-induced vasoconstriction, and antihypertensive effect in SHRs after i.v. injection, but not after oral administration. The toxicity of peptides to reduce the viability of cultured cells was in the millimolar range. In conclusion, we have obtained novel rationally designed heptapeptides with improved ACE inhibitory properties when compared to lead hexapeptides. One selected hexapeptide and two heptapeptides show oral antihypertensive effects in SHRs and appear safe in cytotoxicity assays.
Assuntos
Angiotensina I/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Oligopeptídeos/farmacologia , Peptidil Dipeptidase A/sangue , Células 3T3 , Administração Oral , Angiotensina I/metabolismo , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/uso terapêutico , Artérias Carótidas/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Hipertensão/sangue , Hipertensão/fisiopatologia , Infusões Intravenosas , Masculino , Camundongos , Oligopeptídeos/síntese química , Oligopeptídeos/uso terapêutico , Coelhos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Técnicas de Cultura de Tecidos , Vasoconstrição/efeitos dos fármacosRESUMO
Endothelin-converting enzyme (ECE), a key peptidase in the endothelin (ET) system, cleaves inactive big ET-1 to produce active ET-1, which binds to ET(A) receptors to exert its vasoconstrictor and pressor effects. ECE inhibition could be beneficial in the treatment of hypertension. In this study, a set of eight lactoferricin B (LfcinB)-derived peptides, previously characterized in our laboratory as angiotensin-converting enzyme (ACE) inhibitory peptides, was examined for their inhibitory effects on ECE. In vitro inhibitory effects on ECE activity were assessed using both the synthetic fluorogenic peptide substrate V (FPS V) and the natural substrate big ET-1. To study vasoactive effects, an ex vivo functional assay was developed using isolated rabbit carotid artery segments. With FPS V, only four LfcinB-derived peptides induced inhibition of ECE activity, whereas the eight peptides showed ECE inhibitory effects with big ET-1 as substrate. Regarding the ex vivo assays, six LfcinB-derived peptides showed inhibition of big ET-1-induced, ECE-dependent vasoconstriction. A positive correlation between the inhibitory effects of LfcinB-derived peptides on ECE activity when using big ET-1 and the inhibitory effects on ECE-dependent vasoconstriction was shown. ECE-independent vasoconstriction induced by ET-1 was not affected, thus discarding effects of LfcinB-derived peptides on ET(A) receptors or intracellular signal transduction mechanisms. In conclusion, a combined in vitro and ex vivo method to assess the effects of potentially antihypertensive peptides on the ET system has been developed and applied to show the inhibitory effects on ECE-dependent vasoconstriction of six LfcinB-derived peptides, five of which were dual vasopeptidase (ACE/ECE) inhibitors.
Assuntos
Ácido Aspártico Endopeptidases/farmacologia , Hipertensão/fisiopatologia , Lactoferrina/farmacologia , Metaloendopeptidases/farmacologia , Peptídeos , Vasoconstrição/efeitos dos fármacos , Animais , Ácido Aspártico Endopeptidases/metabolismo , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina , Humanos , Lactoferrina/genética , Lactoferrina/metabolismo , Masculino , Metaloendopeptidases/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Coelhos , Receptor de Endotelina A/metabolismo , Transdução de Sinais/fisiologia , Vasoconstrição/fisiologiaRESUMO
A set of eight lactoferricin B (LfcinB)-derived peptides was examined for inhibitory effects on angiotensin I-converting enzyme (ACE) activity and ACE-dependent vasoconstriction, and their hypotensive effect in spontaneously hypertensive rats (SHR). Peptides were derived from different elongations both at the C-terminal and N-terminal ends of the representative peptide LfcinB(20-25), which is known as the LfcinB antimicrobial core. All of the eight LfcinB-derived peptides showed in vitro inhibitory effects on ACE activity with different IC(50) values. Moreover, seven of them showed ex vivo inhibitory effects on ACE-dependent vasoconstriction. No clear correlation between in vitro and ex vivo inhibitory effects was found. Only LfcinB(20-25) and one of its fragments, F1, generated after a simulated gastrointestinal digestion, showed significant antihypertensive effects in SHR after oral administration. Remarkably, F1 did not show any effect on ACE-dependent vasoconstriction in contrast to the inhibitory effect showed by LfcinB(20-25). In conclusion, two LfcinB-derived peptides lower blood pressure and exhibit potential as orally effective antihypertensive compounds, yet a complete elucidation of the mechanism(s) involved deserves further ongoing research.
Assuntos
Anti-Hipertensivos/administração & dosagem , Hipertensão/tratamento farmacológico , Lactoferrina/administração & dosagem , Peptídeos/administração & dosagem , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/química , Modelos Animais de Doenças , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptidil Dipeptidase A/metabolismo , Coelhos , Ratos , Ratos Endogâmicos SHRRESUMO
Cardiovascular disease is the major cause of morbidity and mortality in diabetic patients, which in turn is also associated with low levels of serum testosterone. The working hypothesis was that diabetes might modify the mechanisms involved in the vascular actions of testosterone in isolated rabbit carotid arteries. Testosterone (10(-8)-3x10(-4)M) induced a concentration-dependent relaxation of precontracted carotid arteries, which was higher in diabetic than in control rabbits. In control rabbits neither endothelium removal nor the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine (l-NOArg, 10(-5)M) modified the relaxant action of testosterone, and the cyclooxygenase (COX) inhibitor indomethacin (10(-5)M) enhanced this relaxation. In contrast, in diabetic rabbits endothelium removal, l-NOArg (10(-5)M) or indomethacin (10(-5)M) inhibited the testosterone induced relaxation. In arteries from diabetic rabbits, eNOS, iNOS and COX-2 expression and testosterone induced release of prostacyclin resulted enhanced in comparison with arteries from control rabbits. Testosterone (10(-4)M) strongly inhibited CaCl(2) (10(-5)-3x10(-2)M) concentration-related contractions of the carotid artery both in control and diabetic rabbits. These results suggest that testosterone relaxes the rabbit carotid artery by blocking the extracellular calcium entry. Diabetes enhances the vasodilator response of the rabbit carotid artery to testosterone by a mechanism that at least includes an increased modulatory activity of the endothelial nitric oxide and an augmented release of COX-2 vasodilator, prostacyclin rather than the absence of COX-1 vasoconstrictor, thromboxane A(2). The hypotestosteronemia observed in diabetic rabbits could be a consequence of the increased expression of iNOS and could contribute to the hyperreactivity of the rabbit carotid artery to testosterone.
Assuntos
Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/etiologia , Ciclo-Oxigenase 2/metabolismo , Diabetes Mellitus Experimental/enzimologia , Angiopatias Diabéticas/etiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Testosterona/metabolismo , Vasodilatação , Animais , Apamina/farmacologia , Glicemia/metabolismo , Western Blotting , Cálcio/metabolismo , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiopatologia , Doenças das Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/fisiopatologia , Charibdotoxina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Epoprostenol/metabolismo , Técnicas Imunoenzimáticas , Indometacina/farmacologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Nitroarginina/farmacologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Coelhos , Tromboxano A2/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
Kidney disease is a frequent complication in diabetes, and significant differences have been reported between male and female patients. Our working hypothesis was that diabetes might modify the vascular actions of testosterone in isolated rabbit renal arteries and the mechanisms involved in these actions. Testosterone (10(-8) to 10(-4)M) induced relaxation of precontracted arteries, without significant differences between control and diabetic rabbits. Both in control and diabetic rabbits endothelium removal inhibited testosterone relaxant action. In arteries with endothelium, incubation with indomethacin (10(-5)M), N(G)-nitro-l-arginine (10(-5)M) or tetraethylammonium (10(-5)M) did not modify relaxations to testosterone neither in control nor in diabetic rabbits. In endothelium-denuded arteries indomethacin enhanced the relaxant action of testosterone, both in control and diabetic rabbits. In arteries from diabetic rabbits, eNOS, iNOS and COX-1 expression and testosterone-induced release of thromboxane A(2) and prostacyclin were not significantly different from those observed in control rabbits. However, COX-2 expression was significantly lower in diabetic rabbits that in control rabbits. In nominally Ca(2+)-free medium, cumulative addition of CaCl2 (10(-5) to 3x10(-2)M) contracted previously depolarized arteries. Testosterone (10(-4)M) inhibited CaCl2 contractions of the renal artery both in control and diabetic rabbits. These results show that testosterone relaxes the renal artery both in control and diabetic rabbits. This relaxation is modulated by muscular thromboxane A(2), it is partially mediated by endothelial prostacyclin, and it involves the blocking of extracellular Ca2+ entry. Diabetes does not modify the mechanisms involved in the relaxant action of testosterone in the rabbit renal artery.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Artéria Renal/metabolismo , Testosterona/metabolismo , Vasodilatação , Animais , Glicemia/metabolismo , Western Blotting , Cálcio/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Técnicas Imunoenzimáticas , Indometacina/farmacologia , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroarginina/farmacologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Prostaglandinas I/metabolismo , Coelhos , Artéria Renal/fisiopatologia , Transdução de Sinais , Testosterona/sangue , Tetraetilamônio/farmacologia , Tromboxano A2/metabolismoRESUMO
A selection of lactoferricin B (LfcinB)-related peptides with an angiotensin I-converting enzyme (ACE) inhibitory effect have been examined using in vitro and ex vivo functional assays. Peptides that were analyzed included a set of sequence-related antimicrobial hexapeptides previously reported and two representative LfcinB-derived peptides. In vitro assays using hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I as substrates allowed us to select two hexapeptides, PACEI32 (Ac-RKWHFW-NH2) and PACEI34 (Ac-RKWLFW-NH2), and also a LfcinB-derived peptide, LfcinB17-31 (Ac-FKCRRWQWRMKKLGA-NH2). Ex vivo functional assays using rabbit carotid arterial segments showed PACEI32 (both D- and L-enantiomers) and LfcinB17-31 have inhibitory effects on ACE-dependent angiotensin I-induced contraction. None of the peptides exhibited in vitro ACE inhibitory activity using bradykinin as the substrate. In conclusion, three bioactive lactoferricin-related peptides exhibit inhibitory effects on both ACE activity and ACE-dependent vasoconstriction with potential to modulate hypertension that deserves further investigation.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Lactoferrina/farmacologia , Peptídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Angiotensina I/farmacologia , Animais , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/fisiologia , Técnicas In Vitro , Lactoferrina/química , Masculino , Peptídeos/química , Peptidil Dipeptidase A/fisiologia , CoelhosRESUMO
BACKGROUND AND PURPOSE: It has been shown that the reversed operation of glutamate transporters when ATP levels fall accounts for most glutamate release induced by severe cerebral ischemia. Nitric oxide (NO) is formed after ischemia and causes ATP depletion. Our purpose is to test if NO release from inducible NO synthase (iNOS) after stroke may cause a delayed glutamate release due to ATP depletion that might underlie progression of the ischemic infarct. We have studied the effect of the highly selective inhibitor of iNOS activity 1400W on brain ATP levels, extracellular glutamate, and stroke outcome after transient focal cerebral ischemia in rats. METHODS: To induce focal ischemia, the middle cerebral artery (MCA) was occluded by using the intraluminal thread technique (tMCAO). 1400W was administered, after tMCAO, by using an Alzet osmotic pump to yield a drug delivery rate of 2.5 mg/kg/h. Results. Postischemic treatment with 1400W induced a reduction in the neurofunctional impairment and in the total volume of brain infarct. Western blot analysis showed ischemia-induced expression of iNOS. Treatment with 1400W partially prevented delayed ATP reduction and produced inhibition of the subsequent delayed increase in glutamate levels caused by the ischemic insult. CONCLUSIONS: Our data indicate that 1400W improves stroke outcome, an effect concomitant to the inhibition of both ischemia-induced decrease in brain ATP levels and increase in glutamate release. These results provide evidence indicating that the expression of iNOS induced by ischemia may contribute to the progression of the ischemic infarct and have important therapeutic implications for the management of stroke.