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1.
Nephrol Dial Transplant ; 38(2): 322-343, 2023 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-35867864

RESUMO

BACKGROUND: In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype. METHODS: This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2-3 studying a relationship between FGF23 and vascular dysfunction. RESULTS: In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2-3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222. CONCLUSION: In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.


Assuntos
MicroRNAs , Insuficiência Renal Crônica , Ratos , Animais , Músculo Liso Vascular , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Análise de Onda de Pulso , Fenótipo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Proliferação de Células
2.
BMC Bioinformatics ; 23(1): 48, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35062867

RESUMO

BACKGROUND: Fluorescence image analysis in biochemical science often involves the complex tasks of identifying samples for analysis and calculating the desired information from the intensity traces. Analyzing giant unilamellar vesicles (GUVs) is one of these tasks. Researchers need to identify many vesicles to statistically analyze the degree of molecular interaction or state of molecular organization on the membranes. This analysis is complicated, requiring a careful manual examination by researchers, so automating the analysis can significantly aid in improving its efficiency and reliability. RESULTS: We developed a convolutional neural network (CNN) assisted intelligent analysis routine based on the whole 3D z-stack images. The programs identify the vesicles with desired morphology and analyzes the data automatically. The programs can perform protein binding analysis on the membranes or state decision analysis of domain phase separation. We also show that the method can easily be applied to similar problems, such as intensity analysis of phase-separated protein droplets. CNN-based classification approach enables the identification of vesicles even from relatively complex samples. We demonstrate that the proposed artificial intelligence-assisted classification can further enhance the accuracy of the analysis close to the performance of manual examination in vesicle selection and vesicle state determination analysis. CONCLUSIONS: We developed a MATLAB based software capable of efficiently analyzing confocal fluorescence image data of giant unilamellar vesicles. The program can automatically identify GUVs with desired morphology and perform intensity-based calculation and state decision for each vesicle. We expect our method of CNN implementation can be expanded and applied to many similar problems in image data analysis.


Assuntos
Inteligência Artificial , Lipossomas Unilamelares , Processamento de Imagem Assistida por Computador , Redes Neurais de Computação , Reprodutibilidade dos Testes
3.
Langmuir ; 38(15): 4702-4712, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35385290

RESUMO

Protein cargos anchored on the lipid membrane can be segregated by fluidic domain phase separation. Lipid membranes at certain compositions may separate into lipid domains to segregate cargos, and protein cargos themselves may be involved in protein condensate domain formation with multivalent binding proteins to segregate cargos. Recent studies suggest that these two driving forces of phase separation closely interact on the lipid membranes to promote codomain formation. In this report, we studied the effect of cargo density on the outcome of the cargo phase separation on giant unilamellar vesicles. Proteins and lipids are connected only by the anchored cargos, so it was originally hypothesized that higher cargo density would increase the degree of interaction between the lipid and protein domains, promoting more phase separation. However, fluorescence image analysis on different cargo densities showed that the cooperative domain formation and steric pressure are at a tug of war opposing each other. Cooperative domain formation is dominant under lower anchor density conditions, and above a threshold density, steric pressure was dominant opposing the domain formation. The result suggests that the cargo density is a key parameter affecting the outcome of cargo organization on the lipid membranes by phase separation.


Assuntos
Microdomínios da Membrana , Lipossomas Unilamelares , Fenômenos Biofísicos , Bicamadas Lipídicas/química , Lipídeos/química , Microdomínios da Membrana/química , Proteínas/metabolismo , Lipossomas Unilamelares/química
4.
IUBMB Life ; 73(7): 900-915, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34033211

RESUMO

Toll-like receptors (TLRs) or pattern recognition receptors respond to pathogen-associated molecular patterns (PAMPs) or internal damage-associated molecular patterns (DAMPs). TLRs are integral membrane proteins with both extracellular leucine-rich and cytoplasmic domains that initiate downstream signaling through kinases by activating transcription factors like AP-1 and NF-κB, which lead to the release of various inflammatory cytokines and immune modulators. In the central nervous system, different TLRs are expressed mainly in microglia and astroglial cells, although some TLRs are also expressed in oligodendroglia and neurons. Activation of TLRs triggers signaling cascades by the host as a defense mechanism against invaders to repair damaged tissue. However, overactivation of TLRs disrupts the sustained immune homeostasis-induced production of pro-inflammatory molecules, such as cytokines, miRNAs, and inflammatory components of extracellular vesicles. These inflammatory mediators can, in turn, induce neuroinflammation, and neural tissue damage associated with many neurodegenerative diseases. This review discusses the critical role of TLRs response in Alzheimer's disease, Parkinson's disease, ischemic stroke, amyotrophic lateral sclerosis, and alcohol-induced brain damage and neurodegeneration.


Assuntos
Alcoolismo/fisiopatologia , Encéfalo/efeitos dos fármacos , Doenças Neurodegenerativas/etiologia , Doenças Neuroinflamatórias/etiologia , Receptores Toll-Like/fisiologia , Alcoolismo/etiologia , Animais , Encéfalo/fisiopatologia , Exossomos/patologia , Exossomos/fisiologia , Expressão Gênica , Humanos , Imunidade Inata , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças Neurodegenerativas/terapia , Doenças Neuroinflamatórias/terapia
5.
Nanomedicine ; 34: 102376, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33667725

RESUMO

Alcohol abuse induces the expression of inflammatory mediators by activating the immune receptors to trigger neuroinflammation and brain damage; however, therapies that reduce neuroimmune system activation may protect against alcohol's damaging effects. Curcuminoids possess anti-inflammatory properties but suffer from low bioavailability; therefore, we designed a new receptor-targeted biodegradable star-shaped crosslinked polypeptide polymer that bears propargylamine moieties and bisdemethoxycurcumin (StClPr-BDMC-ANG) as an enhanced anti-inflammatory therapeutic that penetrates the blood-brain-barrier and ameliorates alcohol-induced neuroinflammation. StClPr-BDMC-ANG administration maintains the viability of primary glia and inhibits the ethanol-induced upregulation of crucial inflammatory mediators in the prefrontal and medial cortex in a mouse model of chronic ethanol consumption. StClPr-BDMC-ANG treatment also suppresses the ethanol-mediated downregulation of microRNAs known to negatively modulate neuroinflammation in the brain cortex (miRs 146a-5p and let-7b-5p). In summary, our results demonstrate the attenuation of alcohol-induced neuroinflammation by an optimized and targeted polypeptide-based nanoconjugate of a curcuminoid.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Curcumina/análogos & derivados , Nanoconjugados/administração & dosagem , Doenças Neuroinflamatórias/tratamento farmacológico , Peptídeos/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Curcumina/administração & dosagem , Curcumina/química , Camundongos , Nanoconjugados/química , Doenças Neuroinflamatórias/induzido quimicamente , Peptídeos/química
6.
Int J Mol Sci ; 22(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921831

RESUMO

Human intestinal microbiota comprise of a dynamic population of bacterial species and other microorganisms with the capacity to interact with the rest of the organism and strongly influence the host during homeostasis and disease. Commensal and pathogenic bacteria coexist in homeostasis with the intestinal epithelium and the gastrointestinal tract's immune system, or GALT (gut-associated lymphoid tissue), of the host. However, a disruption to this homeostasis or dysbiosis by different factors (e.g., stress, diet, use of antibiotics, age, inflammatory processes) can cause brain dysfunction given the communication between the gut and brain. Recently, extracellular vesicles (EVs) derived from bacteria have emerged as possible carriers in gut-brain communication through the interaction of their vesicle components with immune receptors, which lead to neuroinflammatory immune response activation. This review discusses the critical role of bacterial EVs from the gut in the neuropathology of brain dysfunctions by modulating the immune response. These vesicles, which contain harmful bacterial EV contents such as lipopolysaccharide (LPS), peptidoglycans, toxins and nucleic acids, are capable of crossing tissue barriers including the blood-brain barrier and interacting with the immune receptors of glial cells (e.g., Toll-like receptors) to lead to the production of cytokines and inflammatory mediators, which can cause brain impairment and behavioral dysfunctions.


Assuntos
Vesículas Extracelulares/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Microbiota/fisiologia , Neuroblastoma/metabolismo , RNA Longo não Codificante/metabolismo
7.
Cell Physiol Biochem ; 52(1): 76-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790506

RESUMO

BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.


Assuntos
Membrana Celular/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteína Quinase C-alfa/metabolismo , Vasodilatação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Dibutirato de 12,13-Forbol/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Quinases Associadas a rho/metabolismo
8.
Stroke ; 49(6): 1507-1510, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29735721

RESUMO

BACKGROUND AND PURPOSE: Rho-kinase, an effector of RhoA, is associated with various cardiovascular diseases in circulating blood cells. However, the role of RhoA/Rho-kinase in peripheral blood mononuclear cells from patients with spontaneous aneurysmal subarachnoid hemorrhage (aSAH) has not yet been studied in relation to the severity of this disease. Therefore, we analyzed the expression and activity of RhoA as a possible biomarker in aSAH. METHODS: Twenty-four patients with aSAH and 15 healthy subjects were examined. Peripheral blood mononuclear cells were collected, and RhoA activity and expression were determined by RhoA activation assay kit (G-LISA) and enzyme-linked immunosorbent assay tests, respectively. The severity of aSAH was determined from the World Federation of Neurological Surgeon scale, and vasospasm was evaluated using clinical symptoms, arteriography, and sonography. RESULTS: RhoA expression was significantly increased in peripheral blood mononuclear cells from patients on days 0, 2, and 4 after aSAH versus healthy subjects (P=0.036, 0.010, and 0.018, respectively, by U Mann-Whitney analysis). There was a significant correlation between RhoA expression and injury severity on days 2 and 4 (Spearman test, day 2: r=0.682, n=14, P=0.007; day 4: r=0.721, n=14, P=0.004). No significant correlation was observed on day 0 (day 0: r=0.131, n=6, P=0.805). Active RhoA was not significantly different in patients and healthy subjects on days 0, 2, and 4 (P=0.243, 0.222, and 0.600, respectively) nor did it increase significantly on days 0 and 2 in patients with vasospasm versus patients without vasospasm (P=0.064 and 0.519, respectively). In contrast, active RhoA was significantly higher on day 4 in patients who developed vasospasm versus patients without vasospasm (P=0.028). CONCLUSIONS: Our preliminary results indicate that RhoA expression and activity in peripheral blood mononuclear cells might be related with aSAH severity and cerebral vasospasm. RhoA is a potential biomarker of the risks associated with aSAH.


Assuntos
Leucócitos Mononucleares/metabolismo , Hemorragia Subaracnóidea/metabolismo , Vasoespasmo Intracraniano/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Biomarcadores/sangue , Angiografia Cerebral/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/diagnóstico
9.
Vascul Pharmacol ; 155: 107287, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38408532

RESUMO

Aneurismal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by the rupture of the cerebral arteries and the extravasation of blood to the subarachnoid space and is accompanied by severe comorbidities. Secondarily associated vasospasm is one of the main side effects after hydrocephalus and possible rebleeding. Here, we analyze the alterations in function in the arteries of a rat model of SAH. For this, autologous blood was injected into the cisterna magna. We performed electrophysiological, microfluorimetric, and molecular biology experiments at different times after SAH to determine the functional and molecular changes induced by the hemorrhage. Our results confirmed that in SAH animals, arterial myocytes were depolarized on days 5 and 7, had higher [Ca2+]i on baseline, peaks and plateaus, and were more excitable at low levels of depolarization on day 7, than in the control and sham animals. Microarray analysis showed that, on day 7, the sets of genes related to voltage-dependent Ca2+ channels and K+ dynamics in SAH animals decreased, while the voltage-independent Ca2+ dynamics genes were over-represented. In conclusion, after SAH, several mechanisms involved in arterial reactivity were altered in our animal model, suggesting that there is no unique cause of vasospasm and alterations in several signaling pathways are involved in its development.


Assuntos
Modelos Animais de Doenças , Hemorragia Subaracnóidea , Vasoespasmo Intracraniano , Animais , Hemorragia Subaracnóidea/fisiopatologia , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/patologia , Masculino , Vasoespasmo Intracraniano/fisiopatologia , Vasoespasmo Intracraniano/metabolismo , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/patologia , Sinalização do Cálcio , Fatores de Tempo , Artérias Cerebrais/metabolismo , Artérias Cerebrais/fisiopatologia , Artérias Cerebrais/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Músculo Liso Vascular/patologia , Ratos Sprague-Dawley , Regulação da Expressão Gênica , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Ratos
10.
Transl Stroke Res ; 15(2): 378-387, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-36814009

RESUMO

Aneurysmal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by extravasation of blood to the subarachnoid space after rupture of the cerebral vessels. After bleeding, the immune response is activated. The role of peripheral blood mononuclear cells (PBMCs) in this response is a current subject of research. We have analysed the changes in PBMCs of patients with aSAH and their interaction with the endothelium, focusing on their adhesion and the expression of adhesion molecules. Using an in vitro adhesion assay, we observed that the adhesion of PBMCs of patients with aSAH is increased. Flow cytometry analysis shows that monocytes increased significantly in patients, especially in those who developed vasospasm (VSP). In aSAH patients, the expression of CD162, CD49d, CD62L and CD11a in T lymphocytes and of CD62L in monocytes increased. However, the expression of CD162, CD43, and CD11a decreased in monocytes. Furthermore, monocytes from patients who developed arteriographic VSP had lower expression of CD62L. In conclusion, our results confirm that after aSAH, monocyte count and adhesion of PBMCs increase, especially in patients with VSP, and that the expression of several adhesion molecules is altered. These observations can help predict VSP and to improve the treatment of this pathology.


Assuntos
Hemorragia Subaracnóidea , Vasoespasmo Intracraniano , Humanos , Leucócitos Mononucleares , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/etiologia , Monócitos , Angiografia
11.
Circ Res ; 108(11): 1348-57, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21493898

RESUMO

BACKGROUND: Sustained vascular smooth muscle contraction is mediated by extracellular Ca(2+) influx through L-type voltage-gated Ca(2+) channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca(2+) sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca(2+) release from the sarcoplasmic reticulum (calcium channel-induced Ca(2+) release). We have studied the functional role of calcium channel-induced Ca(2+) release and the inter-relations between Ca(2+) channel and RhoA/ROCK activation. METHODS AND RESULTS: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca(2+) and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca(2+) channel activation and subsequent metabotropic Ca(2+) release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca(2+) from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca(2+) release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca(2+) influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. CONCLUSIONS: These findings reveal that calcium channel-induced Ca(2+) release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca(2+) channel activation with metabotropic Ca(2+) release and sensitization (RhoA/ROCK stimulation).


Assuntos
Canais de Cálcio Tipo L/fisiologia , Músculo Liso Vascular/fisiologia , Vasoconstrição/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Artéria Basilar/fisiologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Vasoespasmo Coronário/fisiopatologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Hipertensão/fisiopatologia , Indóis/farmacologia , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Nifedipino/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Coelhos , Ratos , Retículo Sarcoplasmático/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasoespasmo Intracraniano/fisiopatologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP
12.
Front Physiol ; 14: 1142354, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36935756

RESUMO

An adequate supply of oxygen (O2) is essential for most life forms on earth, making the delivery of appropriate levels of O2 to tissues a fundamental physiological challenge. When O2 levels in the alveoli and/or blood are low, compensatory adaptive reflexes are produced that increase the uptake of O2 and its distribution to tissues within a few seconds. This paper analyzes the most important acute vasomotor responses to lack of O2 (hypoxia): hypoxic pulmonary vasoconstriction (HPV) and hypoxic vasodilation (HVD). HPV affects distal pulmonary (resistance) arteries, with its homeostatic role being to divert blood to well ventilated alveoli to thereby optimize the ventilation/perfusion ratio. HVD is produced in most systemic arteries, in particular in the skeletal muscle, coronary, and cerebral circulations, to increase blood supply to poorly oxygenated tissues. Although vasomotor responses to hypoxia are modulated by endothelial factors and autonomic innervation, it is well established that arterial smooth muscle cells contain an acute O2 sensing system capable of detecting changes in O2 tension and to signal membrane ion channels, which in turn regulate cytosolic Ca2+ levels and myocyte contraction. Here, we summarize current knowledge on the nature of O2 sensing and signaling systems underlying acute vasomotor responses to hypoxia. We also discuss similarities and differences existing in O2 sensors and effectors in the various arterial territories.

13.
J Cereb Blood Flow Metab ; 43(11): 1919-1930, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37357772

RESUMO

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease with high morbidity and mortality rates. Within 24 hours after aSAH, monocytes are recruited and enter the subarachnoid space, where they mature into macrophages, increasing the inflammatory response and contributing, along with other factors, to delayed neurological dysfunction and poor outcomes. High-density lipoproteins (HDL) are lipid-protein complexes that exert anti-inflammatory effects but under pathological conditions undergo structural alterations that have been associated with loss of functionality. Plasma HDL were isolated from patients with aSAH and analyzed for their anti-inflammatory activity and protein composition. HDL isolated from patients lost the ability to prevent VCAM-1 expression in endothelial cells (HUVEC) and subsequent adhesion of THP-1 monocytes to the endothelium. Proteomic analysis showed that HDL particles from patients had an altered composition compared to those of healthy subjects. We confirmed by western blot that low levels of apolipoprotein A4 (APOA4) and high of serum amyloid A1 (SAA1) in HDL were associated with the lack of anti-inflammatory function observed in aSAH. Our results indicate that the study of HDL in the pathophysiology of aSAH is needed, and functional HDL supplementation could be considered a novel therapeutic approach to the treatment of the inflammatory response after aSAH.


Assuntos
Hemorragia Subaracnóidea , Humanos , Lipoproteínas HDL , Células Endoteliais/patologia , Proteômica , Anti-Inflamatórios , Proteína Amiloide A Sérica
14.
Circ Res ; 106(7): 1285-9, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20299662

RESUMO

RATIONALE: In arterial myocytes, membrane depolarization-induced Ca(2+) release (DICR) from the sarcoplasmic reticulum (SR) occurs through a metabotropic pathway that leads to inositol trisphosphate synthesis independently of extracellular Ca(2+) influx. Despite the fundamental functional relevance of DICR, its molecular bases are not well known. OBJECTIVE: Biophysical and pharmacological data have suggested that L-type Ca(2+) channels could be the sensors coupling membrane depolarization to SR Ca(2+) release. This hypothesis was tested using smooth muscle-selective conditional Ca(v)1.2 knockout mice. METHODS AND RESULTS: In aortic myocytes, the decrease of Ca(2+) channel density was paralleled by the disappearance of SR Ca(2+) release induced by either depolarization or Ca(2+) channel agonists. Ca(v)1.2 channel deficiency resulted in almost abolition of arterial ring contraction evoked by DICR. Ca(2+) channel-null cells showed unaltered caffeine-induced Ca(2+) release and contraction. CONCLUSION: These data suggest that Ca(v)1.2 channels are indeed voltage sensors coupled to the metabolic cascade, leading to SR Ca(2+) release. These findings support a novel, ion-independent, functional role of L-type Ca(2+) channels linked to intracellular signaling pathways in vascular myocytes.


Assuntos
Canais de Cálcio Tipo L/deficiência , Sinalização do Cálcio , Músculo Liso Vascular/metabolismo , Retículo Sarcoplasmático/metabolismo , Vasoconstrição , Animais , Aorta/metabolismo , Cafeína/farmacologia , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio/efeitos dos fármacos , Potenciais da Membrana , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Potássio/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Sódio/metabolismo , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos
15.
J ISAKOS ; 7(6): 201-205, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35973626

RESUMO

OBJECTIVES: The influence of local antibiotic therapy in orthopedic surgery remains unclear. In this trial, we evaluated the incidence of periprosthetic joint infections (PJI), after local or intravenous (IV) antibiotic prophylaxis. The aim of this intervention was to compare the PJI incidence in a population with non-modifiable risk factors after local prophylaxis with vancomycin-loaded calcium sulfate beads versus a control group. METHODS: A total of 83 subjects were evaluated, inclusion criteria included participants over 60 years of age, with at least one main risk factor for PJI who underwent total hip or knee joint replacement between June 2019 and May 2020. Cases were randomized, and the intervention group received local prophylactic antibiotic therapy with calcium sulfate beads impregnated with vancomycin; conventional IV prophylactic antibiotic therapy was administered for the control group. C-reactive protein (CRP) and erythrocyte sedimentation rate (ERS) serum biomarkers were analyzed on the day 5 and weeks 4, 8, and 12. When needed, the synovial fluid sample was obtained and cultured for the early acute PJI diagnosis. RESULTS: Acute PJI was found in 27 patients (67.5%) in the control group and 4 (9.3%) in the intervention group. The variable analysis identified that local prophylaxis with calcium sulfate beads reduces the incidence of acute knee or hip PJI in patients with non-modifiable risk factors compared to conventional prophylaxis (p < 0.0001) with a relative risk of 0.13 (CI:0.05-0.35). Length of hospital stay was also shorter in the intervention group at 4.6 days, compared to 15.25 days in the control group; p < 0.001. CONCLUSIONS: Local antibiotic prophylaxis in patients with non-modifiable risk factors undergoing hip or knee replacement reduces the incidence of acute PJI compared to IV antibiotics. CLINICAL TRIALS: NCT03976466 (clnicaltrials.gov) LEVEL OF EVIDENCE: II.


Assuntos
Artrite Infecciosa , Artroplastia de Quadril , Infecções Relacionadas à Prótese , Humanos , Pessoa de Meia-Idade , Idoso , Vancomicina/uso terapêutico , Sulfato de Cálcio , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/epidemiologia , Infecções Relacionadas à Prótese/prevenção & controle , Artroplastia de Quadril/efeitos adversos , Incidência , Antibacterianos/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Fatores de Risco
16.
Hypertens Res ; 41(9): 730-737, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30054591

RESUMO

Evidence has shown that vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) are depolarized and that the expression of L-type Ca2+ channels (LTCCs) and the sarcoplasmic reticulum (SR) Ca2+ buffering system are upregulated. Arterial rings exposed to high K+ solutions develop a contraction with two components, namely, an initial or phasic component and a sustained or tonic component. Because LTCCs and SR have different functions in the phasic and tonic components of depolarization-induced contraction, this study investigated the role of LTCC-SR coupling in depolarized arterial rings of SHRs. In the absence of extracellular Ca2+, high external K+ or LTCC agonists elicited a transitory contraction, which was sensitive to nifedipine and was potentiated in SHRs. In the presence of extracellular Ca2+, cyclopiazonic acid (CPA), an SR Ca2+-ATPase (SERCA) inhibitor, evoked a transient contraction that was significantly increased in SHRs. Although the phasic and tonic components were markedly increased in depolarized arterial rings of SHRs, they showed different voltage-dependence and sensitivity to SERCA inhibition. The tonic component was more sensitive to moderate depolarizations, and CPA selectively reduced the tonic component to the level observed in WKY rats. These results suggested that LTCC-SR coupling is potentiated in the sustained contraction of hypertensive VSMCs.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Hipertensão/fisiopatologia , Retículo Sarcoplasmático/fisiologia , Vasoconstrição/fisiologia , Animais , Cálcio/metabolismo , Masculino , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
17.
Cell Calcium ; 42(4-5): 513-20, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17559931

RESUMO

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Artérias/metabolismo , Artérias/fisiologia , Sinalização do Cálcio , Inositol 1,4,5-Trifosfato/metabolismo , Modelos Biológicos , Contração Muscular , Músculo Liso Vascular/fisiologia , Ratos , Fosfolipases Tipo C/metabolismo
18.
Vascul Pharmacol ; 93-95: 33-41, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28526517

RESUMO

The role of L-type Ca2+ channels (LTCCs) and RhoA/Rho kinase (ROCK) on depolarization-induced sustained arterial contraction lasting several minutes is already known. However, in vivo, vascular smooth muscle cells can be depolarized for longer periods, inducing substantial inactivation of LTCCs and markedly reducing Ca2+ influx into the myocytes. We have examined, in femoral arterial rings, the role of LTCCs and RhoA/ROCK during long-lasting depolarization. Our results reveal a new vasoreactive response after 20-30min of depolarization in 2.5mM external Ca2+ that has not been identified previously with shorter stimuli. Prolonged depolarization-induced arterial contraction was permanently abolished when arterial rings were treated with 100nM external Ca2+ or 20nM nifedipine. However, when Ca2+ influx was restricted, applying ~7µM external Ca2+ solution or 3nM nifedipine, vasorelaxation was transient, and isometric force slowly increased after 30min and maintained its level until the end of the stimulus. Under these conditions, arterial contraction showed the same temporal course of RhoA activity and was sensitive to fasudil, nifedipine and cyclopiazonic acid. Ca2+-response curve in ß-escin permeabilized arteries was also sensitive to ROCK inhibitors. Thus, although long-lasting depolarization inactivates LTCCs, the reduced Ca2+ entry can induce a detectable arterial contraction via RhoA/ROCK activation.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Músculo Liso Vascular/enzimologia , Vasoconstrição , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Artéria Femoral/enzimologia , Técnicas In Vitro , Masculino , Potenciais da Membrana , Músculo Liso Vascular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos Wistar , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Vasodilatadores/farmacologia , Quinases Associadas a rho/antagonistas & inibidores
19.
Vascul Pharmacol ; 72: 64-72, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25937251

RESUMO

We have previously described that L-type Ca(2+) channels' (LTCCs) activation and metabotropic Ca(2+) release from the sarcoplasmic reticulum (SR) regulate RhoA/Rho kinase (ROCK) activity and sustained arterial contraction. We have investigated whether this signaling pathway can be altered in a new experimental model of subarachnoid hemorrhage (SAH). For this purpose, arterial reactivity was evaluated on days 1 to 5 after surgery. A significant increase of basal tone, measured 4 and 60min after normalization, was observed on day 5 after SAH and at 60min on days 2 and 3 after SAH. This phenomenon was suppressed with LTCCs and ROCK inhibitors. We have also studied arterial rings vasoreactivity in response to high K(+) solutions. Interestingly, there were no significant differences in the phasic component of the high K(+)-induced contraction between sham and SAH groups, whereas a significant increase in the sustained contraction was observed on day 5 after SAH. This latter component was sensitive to fasudil, and selectively reduced by low nifedipine concentration, and phospholipase C and SR-ATPase inhibitors. Therefore, our data suggest that the metabotropic function of LTCCs is potentiated in SAH. Our results could provide a new strategy to optimize the pharmacological treatment of this pathological process.


Assuntos
Artéria Basilar/metabolismo , Canais de Cálcio Tipo L/metabolismo , Contração Muscular/fisiologia , Retículo Sarcoplasmático/metabolismo , Hemorragia Subaracnóidea/metabolismo , Vasoconstrição/fisiologia , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Artéria Basilar/efeitos dos fármacos , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Nifedipino/farmacologia , Ratos , Ratos Wistar , Retículo Sarcoplasmático/efeitos dos fármacos , Hemorragia Subaracnóidea/tratamento farmacológico , Vasoconstrição/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Cell Calcium ; 36(6): 525-34, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15488602

RESUMO

In smooth muscle cells, oscillations of intracellular Ca2+ concentration ([Ca2+]i) are controlled by inositol 1,4,5-trisphosphate (InsP3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca2+ oscillations are regulated differentially by InsP3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP3 production or activation of InsP3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca2+ free solutions did not prevent the ATP-induced Ca2+ release from InsP3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.


Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Miócitos de Músculo Liso/fisiologia , Artéria Pulmonar/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Condutividade Elétrica , Impedância Elétrica , Técnicas In Vitro , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Coelhos , Rianodina/farmacologia
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